WO1993019089A2 - Oxygen carrier - Google Patents
Oxygen carrier Download PDFInfo
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- WO1993019089A2 WO1993019089A2 PCT/FR1993/000273 FR9300273W WO9319089A2 WO 1993019089 A2 WO1993019089 A2 WO 1993019089A2 FR 9300273 W FR9300273 W FR 9300273W WO 9319089 A2 WO9319089 A2 WO 9319089A2
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- beta
- globin
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 title claims abstract description 47
- 239000001301 oxygen Substances 0.000 title claims abstract description 47
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
- C07K14/805—Haemoglobins; Myoglobins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
Definitions
- the present invention relates to oxygen transporters having a structure similar to hemoglobin and which can be used as a blood substitute having a reduced affinity for oxygen.
- ⁇ is therefore desirable to be able to have an artificial oxygen transporter which can be substituted for blood for transfusion use.
- Hemoglobin is the main constituent of the red blood cell, its essential function is to fix, transport and deliver the quantity of oxygen necessary for the normal functioning of tissues.
- the hemoglobin molecule has two parts, a protein part, globin, and groups that house ferrous iron atoms responsible for fixing oxygen, heme.
- the globin is a tetramer composed of four identical chains two by two which are named, for the peptide chains of 141 amino acids, alpha, and for the chains of 146 amino acids, chains of the beta type; for this reason normal human globin is denoted alpha 2 beta 2 .
- beta chain or “non-alpha” covers not only beta chains, but also chains called epsilon, gamma or delta.
- a hemin group is linked to each of the polypeptide chains.
- hemoglobin Normally in adults, more than 95% of hemoglobin is constituted by alpha 2 beta 2 tetramer, that is to say the association of two heterologous alpha-beta dimers, associated with the catalytic complex, Theme, that is to say, hemoglobin A. There is 2% to 3% of a hemoglobin consisting of tetramers alpha 2 delta 2 , and traces of fetal hemoglobin alpha 2 gamma 2 .
- patent application WO 88/09179 proposed this type of approach, namely the separate synthesis of alpha chains and beta chains, in particular in the form of non-natural mutants.
- the subject of the present invention is an oxygen transporter which requires the synthesis of only one type of chain, this type of chain being able to be associated in the form of a tetramer carrying heme and capable of reversibly fixing the 'oxygen.
- the present invention relates to an oxygen transporter of the type comprising at least four practically identical globin-type polypeptide chains, capable of associating or being associated with one another by incorporating Theme, in order to ensure a binding activity. reversible oxygen.
- polypeptide chains of globin type one refers to chains of alpha type or chains of beta type, and that these chains are practically identical, preferably are completely identical.
- This type of transporter therefore differs from the transporters of the prior art in which two distinct types of chains are always provided which are capable of joining together, such as for example alpha and beta chains, even if, moreover, these chains contain certain modifications or appear as a dimer.
- the oxygen transporter according to the present invention can have two different embodiments.
- the four polypeptide chains are identical and their structure, which borrows from alpha chains and beta-type chains, is such that they will be able to associate by reconstituting an interface similar to the alpha 1 interface, beta 2 which is involved in the association of alpha chains and beta chains of natural HbA.
- the chains are identical and are, in particular, all beta chains which will be associated, in the form of a tetramer.
- the present invention relates to artificial oxygen transporters as described above, comprising at least four polypeptide chains incorporating Theme and each having a sequenced globin type structure:
- - V, X, and Z are globin-like polypeptide sequences
- (W) and (Y) are two complementary polypeptide sequences capable of interacting to constitute an interface which, when it belongs to two distinct chains, is at least in part similar to the alpha 1 beta 2 interface, said chains being able to associate by reconstituting said interface similar to the alpha 1 beta 2 interface in order to ensure a reversible and cooperative binding activity of Toxygen.
- the four polypeptide chains associated in a tetramer are associated "head to tail" so that the comparison of the sequences W 1 of one of the chains with the sequence Y 2 of l the other chain leads, in parallel, to the connection of the sequence Y 1 of the first with the sequence W 2 of the second to reconstitute at least in part the alpha 1 , beta 2 interface of the natural hemoglobin.
- This interface between the alpha chains and the beta chains is involved in the conformational transition responsible in part for the fixing and the release of oxygen, its reconstitution makes it possible to obtain oxygen transporters having satisfactory properties.
- polypeptide chains must have structures which bring them closer to the alpha type chains or the beta type chains.
- X will comprise one or more helical segments chosen from helical segments D, E and F of alpha or beta type chains, said segments may be linked together by non-helical segments or by single bonds.
- the globin V type polypeptide sequence will comprise one or more helical segments chosen from helical segments A, B and C of chains of alpha or beta type, said segments may be linked together by non-helical segments or by single bonds.
- the globin Z type polypeptide sequence will comprise one or more helical segments chosen from helical segments G and H of alpha or beta type chains, said segments may be linked together by non-helical segments or by single bonds.
- the helical segments play an important role in the structure of the normal tetramer and that this type of structure will have to be found in the synthetic tetramer, it is however possible to provide for the association of helical segments of different origin, that is to say either alpha type or beta type, as for the non-helical segments, they can be segments having the structure of their natural counterparts and / or be of different nature even if, moreover , the use of homogeneous sequences any alpha or any beta can be preferred.
- alpha, beta interface as it can be reconstituted by the chains sequenced according to the present invention is preferably the following:
- the different helical segments or not may also include mutations with respect to their natural counterparts, as will be described below.
- mutations preferably relate to the more stable beta-type subunit.
- the following mutations in the beta-type chain can be mentioned more particularly:
- NB only hemoglobins containing a single mutation were taken into account either in the ⁇ subunit or in the ⁇ subunit.
- the W and Y sequences must make it possible to reconstitute an alpha 1 beta 2 interface for HbA or the like which is involved in the transition R—> T. This is why the segments (W) and (Y ) will be similar to the C / CD and FG / G segments of the alpha, beta 2 interface of human hemoglobin, but it is possible to modify it if necessary by predicting certain point mutations.
- the essential elements involved in the structure of the alpha 1 beta 2 interface are as follows:
- polypeptide chains which are more particularly advantageous, mention should be made of the mixed alpha, beta chains, that is to say in particular the chains consisting of the N-terminal end of a beta-type chain of globin, and of the C-terminus of the globin alpha chain; or, conversely, a chain consisting of the N-terminal end of an alpha chain of globin and the C-terminal end of a beta-type chain of globin.
- the alpha-beta junction will preferably be located in the flexible area of the Theme pocket, that is to say in the EF segment.
- the most interesting polypeptide comprises the N-terminal end of the beta chain of globin fused to Tamino-acid 73 included in the C-terminal part of the alpha chain of globin from Tamino-acid 69 included.
- FIG. 8 This type of chimeric molecule associated in tetrameric form is represented in FIG. 8 in which the alpha 1 beta 2 interfaces reconstituted in the tetramer have been visualized.
- two polypeptide chains to be linked by a sequence, preferably peptide, located between the Z sequence of Tune and the V sequence of the other, the two chains are thus integral, which promotes the stability of the tetramer.
- the two chains can also be linked by crosslinking or by any type of biochemical interaction such as, for example, covalent bridging bonds (derivatives of diaspirin or of pyridoxal phosphate).
- the oxygen transporter according to the invention will include a heme structure; it may be a natural heme molecule or a metalioporphyrin in which the iron atom is replaced by another metal, in particular cobalt.
- the tetramer consists essentially of four identical polypeptides each having a beta-globin type structure. This type of tetramer called beta 4 will preferably be constituted by beta type chains comprising one of the mutations mentioned above.
- dimers It could, as has been described, be prepared by association of dimers, these dimers being formed by bonding the N and C ends of two subunits.
- the oxygen transporter according to the invention generally has a reduced affinity for oxygen compared to natural hemoglobin, preferably this affinity is reduced by at least 20% compared to that of natural hemoglobin.
- the transporter according to the present invention is preferably obtained by culture of a transformed organism producing said chain, it may be eukaryotic cells comprising a DNA sequence coding for said chain under the dependence of expression elements ensuring the expression of said sequence in said host cell.
- the host cell is preferably a eukaryotic cell and preferably a yeast and / or a higher eukaryotic cell or a transgenic animal.
- the expression system it may be either plasmids with autonomous replication, or piasmids allowing integration into the genome of the host cell.
- the coding sequences may be dependent on a heterologous but effective promoter in the host or on a homologous promoter, or even on a chromosomal promoter. It is also possible to provide systems ensuring, for example, the secretion of these polypeptide chains.
- tetramers is, most of the time, spontaneous, it can be favored according to the exact structure of the tetramer. by special conditions for dissolving, in particular under certain pH and / or ionic strength conditions.
- hemin group can be spontaneous, in particular in eukaryotes, and the tetramers obtained are "heminized" without there being any need for a specific step. Most of the time Theme will be added at a later stage.
- the present invention also relates to methods allowing the preparation of these polypeptide chains by techniques using the recombinant DNAs and the possible formation of the tetramer, as described above (Hoffman and ⁇ l, 1990; Nagai & Thogersen, 1984, 1987; Nagai and ⁇ l, 1985; Tame and ⁇ l, 1991; Wagenbach and ⁇ l, 1991).
- the present invention relates to the use of chains as a medicament, in particular for the transport of oxygen in combination with an acceptable support in the transfusion field.
- FIG. 1 shows schematically the nucleotide and amino acid sequence of the alpha chain
- FIG. 2 schematizes the nucleotide and amino acid sequence of the beta chain
- FIG. 3 shows schematically the spatial structure of the beta chain, with indication of the nomenclature of the propellers,
- FIG. 4 represents the diagram of the plasmid pATPrTet
- FIG. 5 represents the diagram of the plasmid pATPr cIIFX beta
- FIGS. 6 and 7 show diagrammatically the preparation of the vector pATPr cIIFX beta Gb
- FIG. 8 represents the diagram of the spatial structure of a dimer according to the invention, in particular the reconstruction of the alpha 1 beta 2 interface.
- FIG. 9 schematizes the stages of the construction of the vector pATPr Chim beta73-alpha69.
- the spatial configuration of the beta chain is represented in FIG. 3 with the nomenclature of the various segments which go from the NH 2 terminal end towards the COOH terminal end:
- NA A, AB, B, C, CE, E, EF, F, FG, G, GH, H and HC.
- the segments A, B, C, E, F, G and H have a substantially helical structure while the junction segments do not have such a structure.
- Amino acids are numbered either from the N-terminal end in ascending order, or by assigning an amino acid the designation of its helix and its rank in it.
- the 36th amino acid in the beta chain is Proline, it is beta 36 (C2) Pro, i.e. the second amino acid in Thélice C.
- the basic vector pAT Pr Tet has an origin of replication derived from pBR 322 and carries the gene for resistance to tetracycline. It allows the expression of heterologous proteins under the control of the Pr promoter of phage lambda. The activity of this promoter is repressed by the repressor CI (ailwash CI 8 57 , thermosensitive). The Pr promoter is blocked at 30 ° C. Raising the temperature to 42 ° C inactivates the repressor and induces transcription from the Pr promoter (FIG. 4).
- the E. coli CAG 1139 bacteria carrying the plasmid are cultured at 37 ° C. (1 L LB medium) until an OD at 600 nm of approximately 0.4 is obtained. Chloramphenicol is added to a final concentration of 170 ⁇ g / ml, and the incubation is continued at 37 ° C for 12-16 hours. The bacteria are lysed by treatment with a NaOH-SDS solution, and the plasmid DNA is purified from the bacterial pellet by precipitation with polyethylene giycol. The protocol used is very exactly that described by Sambrook et al (1989).
- the sequence coding for the fusion protein was obtained by the PCR technique from the double-stranded replicative form of phage M 13 mp10 (10 ng).
- the double-stranded replicative form is prepared according to the technique indicated above for the plasmid DNA, from an E. coli bacterial pellet, strain JM 101.
- a site recognized by SnaBI restriction tendonuclease is introduced at the end 5 'of the coding sequence.
- Xhol 25 units, 2 hours, 37 ° C
- It is precipitated with tethanol after extraction with phenol / chloroform.
- the precipitate is taken up in 10 ⁇ l of 10 mM Tris-HCl / 1 mM EDTA buffer.
- the insertion into the expression vector is carried out using a vector / insert ratio of 1/5 (i.e. 3 ⁇ l of the vector DNA solution and 3 ⁇ l of the insert solution).
- the mixture is incubated in the presence of T4 DNA ligase, 4 hours at room temperature.
- the reaction mixture is used to transform competent CAG 1139 bacteria (CaCl 2 treatment).
- the transformed bacteria selected by the resistance to tetracycline are tested for the expression of the fusion protein.
- the vector thus obtained pAT Pr cIIFX beta directs the synthesis of a fusion protein cIIFX beta globin containing:
- the E. coli CAG 1139 strain carrying the plasmid pAT Pr cIIFX beta is incubated at 30 ° C in M9 medium (Na 2 HPO 4 50 mM / KH 2 PO 4 20 mM / NaCI 8.5 mM / NH 4 Cl20 mM / MgSO ⁇ O.lmM) containing 2 g of glucose, 20 g of yeast extract and 0.5 g of vitamin B1 per liter) in the presence of tetracycline (10 mg per liter).
- M9 medium Na 2 HPO 4 50 mM / KH 2 PO 4 20 mM / NaCI 8.5 mM / NH 4 Cl20 mM / MgSO ⁇ O.lmM
- tetracycline 10 mg per liter
- the bacterial pellet (100 g wet weight) is thawed and suspended in 100 ml 50 mM Tris-HCl, pH 8/25% sucrose (w / v) / 1 mM EDTA.
- the bacteria are lysed by incubation in the presence of 200 mg of lysozyme (30 minutes in ice).
- MgCl 2 , MnCl 2 and DNase I are added so as to obtain respective final concentrations of 10 mM, 1 mM and 10 ⁇ g / ml.
- the protein precipitate is dissolved in a solution of guanidine-HCl 6M / Tris-AcOH 25 mM, pH 5.0 / EDTA 1 mM / dithiothreitol 1 mM.
- the solution is equilibrated in an 8M urea buffer / 25 mM Tris-AcOH, pH 5.0 / 1 mM EDTA / 1 mM dithiothreitol by filtration through a column of Sephadex G-25, then deposited on a column of CM-Sepharose (4 ⁇ 10 cm) balanced with the same buffer.
- the fusion protein is eluted using a linear gradient formed by 500 ml of 8M urea / 25 mM Tris-AcOH, pH 5.0 / EDTA 1 mM / dithiothreitol 1 mM and 500 ml of the same buffer supplemented with NaCl qsp a concentration of 0.2 M.
- the fusion protein is then purified by filtration on a column of Sephacryl S-200 (5 ⁇ 60 cm) in guanidine-5 M HCl / 50 mM Tris-HCl, pH 8.0 / 1 mM EDTA / 1 mM dithiothreitol.
- the protein-containing fractions are pooled, dialyzed against water and lyophilized, or dialyzed against the cleavage buffer to be immediately treated with activated factor X.
- the mutation is introduced into TADN using a synthetic oligonucleotide initiator of sequence dC AGG AGT CAG CAT CAC CCT ACC C.
- This sequence is complementary to that of cDNA coding for the beta subunit of globin framing the codon of Thistidine beta (NA2) .
- the triplet introduced CAT (instead of GTG) is altered so as to code for a methionine. Mutagenesis is performed using the replication of the phage M 13 into which is inserted cDNA coding for the beta chain of globin (Nagai & Thogersen, 1984), by the method of Nakamaye & Eckstein (19S6) recommended by Amersham (Kit Amersham).
- the product of the mutagenesis reaction is used to transform competent E. coli bacteria, strain JM 101 according to conventional methods (Nagai & Thogersen, 1987; Sambrook et al, 1989).
- the phages carrying the mutation are detected by T DNA sequencing (Sanger method, 1977).
- the complete sequence of cDNA coding for the fusion protein is verified in order to ensure the integrity of the coding sequence.
- the coding sequence containing the mutation is processed as described above to be inserted into the expression vector.
- the mutation is introduced into cDNA using Toligonuciéotide initiator dAGG AGT CAG GAT CAC CCT ACC C.
- the triplet GAT (for GTG) makes it possible to code for an isoieucine.
- the mutation is introduced into cDNA using Tohgonucleotide initiator dGGA CTC AAA GTA CCT CTG GGT.
- the triplet GTA (for GAA) makes it possible to code for a tyrosine.
- the construction coding for the beta-alpha globin chimera is constructed by PCR from two plasmids:
- Nsi 5 'GCA TTT AAT GCA TTG ATG CC 3'
- the fragment coding for CIIFX NH- (1-73) beta is amplified from the vector pAT Pr beta using the primers Nsi and Dinv.
- the conditions are as follows:
- the fragment coding for alpha (69-141) COOH of alpha globin is amplified from the plasmid alphalpJW101 (Wilson and ⁇ l, 1978) using primers D and Ch.
- the conditions are as follows: 5 ⁇ l PCR buffer
- a third amplification is carried out from the two purified fragments using the primers Nsi and Ch.
- the conditions are as follows:
- PCR product is precipitated with tethanol after extraction with phenol / chloroform, then purified. It is inserted into the plasmid pAT Pr Tet at the Nsi 1 and Xho 1 sites.
- This vector makes it possible to express a fusion protein comprising:
- FIG. 9 shows schematically the stages of construction.
- This plasmid is expressed in E. coli, strain CAG 1139 (Grossman et ⁇ l, 1983, Cell, 32, 1 51-159).
- This phenotype strain: F- thi leu thr LacY TonA SupE galK Lon 100 is transformed by the conventional method of treatment with CaCl 2 .
- the culture is carried out in LB medium in the presence of tetracycline 50 ⁇ g / ml at 30 ° C; when the OD reaches 0.8-1.2, the expression of the DNA protein is induced by passage at 42 ° C. After 2 hours, it is possible to demonstrate the synthesis of the chain (beta-alpha ) on SDS-PAGE gel stained with Coomassie blue by comparison with an untransformed CAG 1139 culture sample. It is estimated that the amount of beta-alpha chain reaches 20% of total proteins. The construction was sequenced to verify that there was no mutation during the successive PCRs. EXAMPLE 9 Functional Characterization of the Recombinant Hemoglobins
- the absorbance spectra are recorded and compared to the spectrum of native oxygenated THb A (Cary 219 spectrophotometer, Varian, Palo Alto, Ca, USA). These spectra make it possible to test the presence of methemoglobin or of hemichrome (Szebeni et al,
- the deoxygenation curves are recorded in solutions of Hb (60 to 100 ⁇ M in heme) in a 50 mM bisTris buffer, 100 mM NaCl, pH 7.2 at 25 ° C., 50 ⁇ M of EDTA and 20 ⁇ g / ml of cataiase are added to the buffer to limit the oxidation of hemoglobin.
- the concentration of NaCl, the pH can be modified to test the different properties of recombinant Hb (Bohr effect, anionic effectors).
- Other natural (2,3 diphosphoglycerate) or non-natural (IHP, clofibrate derivatives) effectors can be added to the solution at the start of the recording.
- the solution is first balanced under a gas mixture whose partial oxygen pressure is at least 350-450 mmHg to ensure an almost complete initial saturation of the hemoglobin.
- the solution is then slowly deoxygenated by admitting pure nitrogen into the measuring cuvette.
- the duration of the recording is 45 to 60 min to ensure the equilibrium conditions. These equilibrium conditions are demonstrated by the superposition of the deoxygenation and re-oxygenation curves.
- the amount of methemoglobin (oxidized Hb) formed during the recording is measured by spectrophotometry according to the method of Benippoh et ai (1973).
- the auto-oxidation speed of hemoglobin is measured by spectrophotometry of the solution (1 00 ⁇ M heme) incubated in a 100 mM Phosphate buffer at 37 ° C. as a function of time (0-> 24 h).
- the thermal stability is measured by incubation of the solutions (300 ⁇ M heme) at 65 ° C as a function of time (0—> 120 min).
- Photodissociation studies are performed using a 10-ns laser system (Quaritel YG 585) delivering 160 mJ at 532 nm.
- the energy of the photodissociation beam can be modified to study the conditions of partial photolysis.
- the samples are studied in cuvettes with 1 or 2 mm optical path.
- the detection beam at 436 nm is almost co-linear with the photodissociation beam.
- the intensity of transmitted light is detected by a photomultipiifier (lP28 Hamamatsu, Japan) and recorded on a digital oscilloscope (Lècroy 9400).
- the data is then transferred to an IBM microcomputer for calculations of absorbance changes and data analysis.
- Kinetic simulations are based on the two-state allosteric model.
- the curve recorded for native Hb A shows the existence of two rates of recombination of CO to hemoglobin: Tune is rapid and corresponds to recombination in the R state of high affinity for the ligand; the second is slower and corresponds to recombination in the T state of low affinity.
- Kinetic experiments make it possible to very quickly test the functionality of recombinant Hb with the minimum of sample.
- the chimeric subunits obtained according to the examples assemble in a tetramer which fixes the oxygen in a reversible manner practically without cooperativity but with an affinity slightly lower than that observed for native Hb A and thirty times lower to that of the ⁇ 4 tetramer.
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Abstract
Description
TRANSPORTEUR D'OXYGENE. OXYGEN CONVEYOR.
La présente invention concerne des transporteurs d'oxygène ayant une structure similaire à l'hémoglobine et qui peuvent être utilisés comme substitut sanguin possédant une affinité réduite pour l'oxygène. The present invention relates to oxygen transporters having a structure similar to hemoglobin and which can be used as a blood substitute having a reduced affinity for oxygen.
La médecine d'urgence requiert de grandes quantités de sang en vue de la transfusion. Outre le problème d'approvisionnement et de stockage pouvant se poser, les transfusions font courir des risques de transmission de maladies infectieuses identifiées ou non et nécessitent, par là même, l'utilisation de procédés d'inactivation virale toujours délicats à mettre en oeuvre sur des protéines. Emergency medicine requires large amounts of blood for transfusion. In addition to the supply and storage problem that may arise, transfusions run the risk of transmitting infectious diseases, whether identified or not, and therefore require the use of viral inactivation processes which are always difficult to implement on proteins.
π est donc souhaitable de pouvoir disposer d'un transporteur d'oxygène artificiel pouvant être substitué au sang dans un usage transfusionnel. π is therefore desirable to be able to have an artificial oxygen transporter which can be substituted for blood for transfusion use.
C'est l'objet de la présente invention qui propose des transporteurs d'oxygène ayant des structures proches de celle de l'hémoglobine humaine (HbA). It is the object of the present invention which provides oxygen transporters having structures close to that of human hemoglobin (HbA).
L'hémoglobine est le constituant principal du globule rouge, elle a pour fonction essentielle de fixer, transporter et délivrer la quantité d'oxygène nécessaire au fonctionnement normal des tissus. Hemoglobin is the main constituent of the red blood cell, its essential function is to fix, transport and deliver the quantity of oxygen necessary for the normal functioning of tissues.
La molécule d'hémoglobine comporte deux parties, une partie protéique, la globine, et des groupements qui abritent des atomes de fer ferreux responsables de la fixation de l'oxygène, l'hème. La globine est un tétramère composé de quatre chaînes identiques deux à deux qui sont nommées, pour les chaînes peptidiques de 141 amino-acides, alpha, et pour les chaînes de 146 amino-acides, chaînes de type bêta ; pour cette raison la globine humaine normale est notée alpha2 bêta2. The hemoglobin molecule has two parts, a protein part, globin, and groups that house ferrous iron atoms responsible for fixing oxygen, heme. The globin is a tetramer composed of four identical chains two by two which are named, for the peptide chains of 141 amino acids, alpha, and for the chains of 146 amino acids, chains of the beta type; for this reason normal human globin is denoted alpha 2 beta 2 .
Le terme de chaîne de type bêta, ou "non alpha", recouvre, non seulement les chaînes bêta, mais également les chaînes dénommées epsilon, gamma ou delta. The term beta chain, or "non-alpha", covers not only beta chains, but also chains called epsilon, gamma or delta.
A chacune des chaînes polypeptidiques est lié un groupe héminique. A hemin group is linked to each of the polypeptide chains.
Normalement chez l'adulte, plus de 95 % de l'hémoglobine sont constitués par du tétramère alpha2 bêta2, c'est-à-dire l'association de deux dimères hétérologues alpha-bêta, associés au complexe catalytique, Thème, c'est-à-dire, l'hémoglobine A. On trouve 2 % à 3 % d'une hémoglobine constituée de tétramères alpha2 delta 2, et des traces d'hémoglobine foetale alpha2 gamma2. Normally in adults, more than 95% of hemoglobin is constituted by alpha 2 beta 2 tetramer, that is to say the association of two heterologous alpha-beta dimers, associated with the catalytic complex, Theme, that is to say, hemoglobin A. There is 2% to 3% of a hemoglobin consisting of tetramers alpha 2 delta 2 , and traces of fetal hemoglobin alpha 2 gamma 2 .
La globine étant constituée par des chaînes protéiques, il était tentant d'essayer de préparer ces chaînes par des techniques mettant en oeuvre les ADN recombinants. As globin is made up of protein chains, it was tempting to try to prepare these chains by techniques using recombinant DNAs.
Les techniques mises en oeuvre ont eu essentiellement pour but de faire synthétiser, par des organismes divers, les chaînes alpha et bêta, ce qui conduit à des procédés lourds d'expression séparée de chacune des chaînes ou des procédés mettant en oeuvre des systèmes de co-expression dont les résultats sont souvent peu satisfaisants. The techniques used were essentially aimed at synthesizing, by various organisms, the alpha and beta chains, which leads to cumbersome processes of separate expression of each of the chains or processes implementing co systems. -expression whose results are often unsatisfactory.
Ainsi, la demande de brevet WO 88/09179 a proposé ce type d'approche, à savoir la synthèse séparée des chaînes alpha et des chaînes bêta, en particulier sous forme de mutants non naturels. Thus, patent application WO 88/09179 proposed this type of approach, namely the separate synthesis of alpha chains and beta chains, in particular in the form of non-natural mutants.
L'approche s'étant révélée relativement lourde, une demande de brevet plus récente, WO 90/13645, a proposé d'utiliser directement un dimère notamment de chaînes alpha destinées à être combinées avec des chaînes bêta. The approach having proved relatively cumbersome, a more recent patent application, WO 90/13645, has proposed to use a dimer directly, in particular of alpha chains intended to be combined with beta chains.
Bien que cette approche soit intéressante, elle nécessite, là encore, l'obtention séparée de séquences alpha et de séquences bêta qui doivent être recombinées, même si celles-ci peuvent se présenter chacune sous forme de dimère afin de permettre la formation d'un tétramère stable. Although this approach is advantageous, it again requires the separate production of alpha and beta sequences which must be recombined, even if these can each be in the form of a dimer in order to allow the formation of a stable tetramer.
La présente invention a pour objet un transporteur d'oxygène qui ne nécessite la synthèse que d'un seul type de chaîne, ce type de chaîne pouvant être associé sous forme d'un tétramère portant l'hème et capable de fixer de façon réversible l'oxygène. The subject of the present invention is an oxygen transporter which requires the synthesis of only one type of chain, this type of chain being able to be associated in the form of a tetramer carrying heme and capable of reversibly fixing the 'oxygen.
Plus particulièrement, la présente invention concerne un transporteur d'oxygène du type comportant au moins quatre chaînes polypeptidiques de type globine pratiquement identiques, susceptibles de s'associer ou d'être associées entre elles en incorporant Thème, afin d'assurer une activité de fixation réversible de l'oxygène. Dans la définition précédente, il faut entendre que par la terminologie "chaînes polypeptidiques de type globine" on se réfère aux chaînes de type alpha ou aux chaînes de type bêta, et que ces chaînes sont pratiquement identiques, de préférence sont totalement identiques. More particularly, the present invention relates to an oxygen transporter of the type comprising at least four practically identical globin-type polypeptide chains, capable of associating or being associated with one another by incorporating Theme, in order to ensure a binding activity. reversible oxygen. In the preceding definition, it should be understood that by the terminology “polypeptide chains of globin type” one refers to chains of alpha type or chains of beta type, and that these chains are practically identical, preferably are completely identical.
Ce type de transporteur se distingue donc des transporteurs de la technique antérieure dans lesquels on prévoit toujours deux types de chaînes distinctes qui sont susceptibles de s'associer, comme par exemple des chaînes alpha et bêta, même si, au demeurant, ces chaînes comportent certaines modifications ou se présentent sous forme de dimère. This type of transporter therefore differs from the transporters of the prior art in which two distinct types of chains are always provided which are capable of joining together, such as for example alpha and beta chains, even if, moreover, these chains contain certain modifications or appear as a dimer.
Le transporteur d'oxygène selon la présente invention peut présenter deux modes de réalisation différents. The oxygen transporter according to the present invention can have two different embodiments.
Dans un premier mode de réalisation, les quatre chaînes polypeptidiques sont identiques et leur structure qui emprunte aux chaînes alpha et aux chaînes de type bêta, est telle qu'elles vont pouvoir s'associer en reconstituant une interface similaire à l'interface alpha1, bêta2 qui intervient dans l'association des chaînes alpha et des chaînes bêta de l'HbA naturelle. In a first embodiment, the four polypeptide chains are identical and their structure, which borrows from alpha chains and beta-type chains, is such that they will be able to associate by reconstituting an interface similar to the alpha 1 interface, beta 2 which is involved in the association of alpha chains and beta chains of natural HbA.
Dans un second mode de réalisation, les chaînes sont identiques et sont, en particulier, toutes des chaînes bêta qui vont être associées, sous forme d'un tétramère. In a second embodiment, the chains are identical and are, in particular, all beta chains which will be associated, in the form of a tetramer.
Plus particulièrement, dans le premier mode de réalisation, la présente invention concerne des transporteurs d'oxygène artificiels tels que décrits précédemment, comportant au moins quatre chaînes polypeptidiques incorporant Thème et ayant chacune une structure de type globine séquencée : More particularly, in the first embodiment, the present invention relates to artificial oxygen transporters as described above, comprising at least four polypeptide chains incorporating Theme and each having a sequenced globin type structure:
V - (W) - X - (Y) - Z V - (W) - X - (Y) - Z
dans laquelle : in which :
- V, X, et Z sont des séquences polypeptidiques de type globine, et - V, X, and Z are globin-like polypeptide sequences, and
- (W) et (Y) sont deux séquences polypeptidiques complémentaires susceptibles d'interagir pour constituer une interface qui, lorsqu'el le appartient à deux chaînes disctinctes, est au moins en partie similaire à l'interface alpha 1 bêta2, iesdites chaînes pouvant s'associer en reconstituant ladite interface similaire à l'interface alpha 1 bêta2 afin d'assurer une activité de fixation réversible et coopérative de Toxygène. - (W) and (Y) are two complementary polypeptide sequences capable of interacting to constitute an interface which, when it belongs to two distinct chains, is at least in part similar to the alpha 1 beta 2 interface, said chains being able to associate by reconstituting said interface similar to the alpha 1 beta 2 interface in order to ensure a reversible and cooperative binding activity of Toxygen.
Dans ce type de transporteur d'oxygène selon l'invention, les quatre chaînes polypeptidiques associées dans un tétramère, sont associées "tête-bêche" afin que la mise en regard des séquences W 1 de Tune des chaînes avec la séquence Y2 de l'autre chaîne conduise, parallèlement, à la mise en rapport de la séquence Y 1 de la première avec la séquence W2 de la seconde pour reconstituer au moins en partie l'interface alpha1 , bêta2 de l'hémoglobine naturelle. In this type of oxygen transporter according to the invention, the four polypeptide chains associated in a tetramer, are associated "head to tail" so that the comparison of the sequences W 1 of one of the chains with the sequence Y 2 of l the other chain leads, in parallel, to the connection of the sequence Y 1 of the first with the sequence W 2 of the second to reconstitute at least in part the alpha 1 , beta 2 interface of the natural hemoglobin.
Cette interface entre les chaînes alpha et les chaînes bêta est impliquée dans la transition conformationnelle responsable en partie de la fixation et de la libération de l'oxygène, sa reconstitution permet d'obtenir des transporteurs d'oxygène ayant des propriétés satisfaisantes. This interface between the alpha chains and the beta chains is involved in the conformational transition responsible in part for the fixing and the release of oxygen, its reconstitution makes it possible to obtain oxygen transporters having satisfactory properties.
Bien entendu, les chaînes polypeptidiques devront présenter des structures qui les rapprochent des chaînes de type alpha ou des chaînes de type bêta. Of course, the polypeptide chains must have structures which bring them closer to the alpha type chains or the beta type chains.
Ainsi, notamment, la séquence poiypeptidique de type globine Thus, in particular, the globin-like polypeptide sequence
X comportera un ou plusieurs segments hélicoïdaux choisis parmi les segments hélicoïdaux D, E et F des chaînes de type alpha ou bêta, lesdits segments pourront être reliés entre eux par des segments non hélicoïdaux ou par des liaisons simples. X will comprise one or more helical segments chosen from helical segments D, E and F of alpha or beta type chains, said segments may be linked together by non-helical segments or by single bonds.
La séquence poiypeptidique de type globine V comportera un ou plusieurs segments hélicoïdaux choisis parmi les segments hélicoïdaux A, B et C des chaînes de type alpha ou bêta, lesdits segments pourront être reliés entre eux par des segments non hélicoïdaux ou par des liaisons simples. The globin V type polypeptide sequence will comprise one or more helical segments chosen from helical segments A, B and C of chains of alpha or beta type, said segments may be linked together by non-helical segments or by single bonds.
La séquence poiypeptidique de type globine Z comportera un ou plusieurs segments hélicoïdaux choisis parmi les segments hélicoïdaux G et H des chaînes de type alpha ou bêta, iesdits segments pourront être reliés entre eux par des segments non hélicoïdaux ou par des liaisons simples. Mais, s'il est clair que les segments hélicoïdaux jouent un rôle important dans la structure du tétramère normal et qu'il faudra retrouver ce type de structure dans le tétramère synthétique, il est toutefois possible de prévoir l'association de segments hélicoïdaux d'origine différente, c'est-à-dire soit de type alpha, soit de type bêta, quant aux segments non hélicoïdaux, ils peuvent être des segments ayant la structure de leurs homologues naturels et/ou être de nature différente même si, au demeurant, l'utilisation de séquences homogènes tout alpha ou tout bêta peut être préférée. The globin Z type polypeptide sequence will comprise one or more helical segments chosen from helical segments G and H of alpha or beta type chains, said segments may be linked together by non-helical segments or by single bonds. However, while it is clear that the helical segments play an important role in the structure of the normal tetramer and that this type of structure will have to be found in the synthetic tetramer, it is however possible to provide for the association of helical segments of different origin, that is to say either alpha type or beta type, as for the non-helical segments, they can be segments having the structure of their natural counterparts and / or be of different nature even if, moreover , the use of homogeneous sequences any alpha or any beta can be preferred.
De façon préférentielle, les segments de la formule générale seront choisis de la manière suivante : Preferably, the segments of the general formula will be chosen as follows:
V alpha NA + A + AB + B + C V alpha NA + A + AB + B + C
W alpha CE (interface alpha 1 /bêta2) W alpha CE (alpha 1 / beta 2 interface)
X alpha E + EF + F X alpha E + EF + F
Y alpha FG (interface alpha 1 /bêta2) Y alpha FG (alpha 1 / beta 2 interface)
Z alpha G + GH + H + HC Z alpha G + GH + H + HC
V bêta NA + A + B + C V beta NA + A + B + C
W bêta CD (interface alpha 1 /bêta2) W beta CD (alpha 1 / beta 2 interface)
X bêta D + E + EF + F X beta D + E + EF + F
Y bêta FG (interface alpha 1 /bêta2) Y beta FG (alpha 1 / beta 2 interface)
Z bêta G + GH + H HC Z beta G + GH + H HC
Enfin, l'interface alpha , bêta- telle qu'elle peut être reconstituée par les chaînes sequencees selon la présente invention est de préférence la suivante : Finally, the alpha, beta interface as it can be reconstituted by the chains sequenced according to the present invention is preferably the following:
( C2 36Pro Val96 G3 ) (C2 36Pro Val96 G3)
( C3 37Trp Pro95 G2 ) (C3 37Trp Pro95 G2)
W(béta-alpha)1 ( C6 40Arg Asp91 G1 ) Y(bêta-alpha)2 W (beta-alpha) 1 (C6 40Arg Asp91 G1) Y (beta-alpha) 2
( C7 41Phe Val93 FG5) (C7 41Phe Val93 FG5)
Arg92 FG4) Arg92 FG4)
Leu91 FG3) ( FG3 91Leu Leu91 FG3) (FG3 91Leu
( FG4 92Arg (FG4 92Arg
( FG5 93 Val Phe41 C7 ) (FG5 93 Val Phe41 C7)
Y(bêta-alpha) 1 ( G1 94Asp Arg40 C6 ) W(bêta-alpha)2 Y (beta-alpha) 1 (G1 94Asp Arg40 C6) W (beta-alpha) 2
( G2 95Pro Trp37 C3 ) (G2 95Pro Trp37 C3)
( G3 96Val Pro36 C2 ) (G3 96Val Pro36 C2)
En outre, les différents segments hélicoïdaux ou non pourront également comporter des mutations par rapport à leurs homologues naturels, comme cela sera décrit ci-après. In addition, the different helical segments or not may also include mutations with respect to their natural counterparts, as will be described below.
Ces mutations concernent de manière préférée la sous-unité de type bêta, plus stable. On peut citer plus particulièrement les mutations suivantes de la chaîne de type bêta : These mutations preferably relate to the more stable beta-type subunit. The following mutations in the beta-type chain can be mentioned more particularly:
- remplacement de His en position 2 par Met - replacement of His in position 2 by Met
- remplacement de Val en position 23 par Ile (Pagnier et αl, 1990) - remplacement de Ala en position 27 par Ser (Baklouti et αl, 1986 ; Fessas et αl, 1982 ; Huang et αl, 1990) - replacement of Val in position 23 by Ile (Pagnier and αl, 1990) - replacement of Ala in position 27 by Ser (Baklouti and αl, 1986; Fessas and αl, 1982; Huang and αl, 1990)
- remplacement de Phe en position 41 par Tyr - replacement of Phe in position 41 by Tyr
- remplacement de His en position 63 par Phe (Lin et αl, 1990 ; Nagai et αl, 1987, 1988) - replacement of His in position 63 by Phe (Lin and αl, 1990; Nagai and αl, 1987, 1988)
- remplacement de His en position 63 par Ile (Lin et ai, 1990 ; Nagai et αl, 1987, 1988) - replacement of His in position 63 by Ile (Lin et ai, 1990; Nagai and αl, 1987, 1988)
- remplacement de Val en position 67 par Phe (Lin et al, 1990 ; Nagai et αl, 1987, 1988) - replacement of Val in position 67 by Phe (Lin et al, 1990; Nagai and αl, 1987, 1988)
- remplacement de Val en position 67 par Ile (Lin et αl, 1990 ; Nagai et al, 1987, 1988) - replacement of Val in position 67 by Ile (Lin and αl, 1990; Nagai et al, 1987, 1988)
De telles mutations permettent de diminuer l'affinité intrinsèque du transporteur pour l'oxygène, tout en maintenant un effet de coopérativité. Such mutations make it possible to reduce the intrinsic affinity of the transporter for oxygen, while maintaining a cooperative effect.
D'autres mutations ont également été décrites et peuvent très bien être incorporées dans les hémoglobines selon la présente invention. Il peut s'agir notamment de Tune ou de plusieurs des mutations figurant au tableau I ci-après. Tableau 1 Other mutations have also been described and may very well be incorporated into the hemoglobins according to the present invention. It may in particular be one or more of the mutations appearing in Table I below. Table 1
Variants naturels de l'Hb humaine présentant une diminution de l'affinité pour l'oxygène Natural variants of human Hb with a reduced affinity for oxygen
Mutants de la chaîne α Mutants of the α chain
Hémoglobine mutation Références Hemoglobin mutation References
Torino α43 (CE1) Phe→ Val Beretta et al, 1968 Torino α43 (CE1) Phe → Val Beretta et al, 1968
Hirosaki α43 (CE1) Phe→ Leu Ohba et al, 1975a Hirosaki α43 (CE1) Phe → Leu Ohba et al, 1975a
Moabit α86 (F7) Leu→ Arg Knuth et αl, 1979 Moabit α86 (F7) Leu → Arg Knuth and αl, 1979
Sétif α94 (G1) Asp→ Tyr Wajcman et αl, 1972 Setif α94 (G1) Asp → Tyr Wajcman and αl, 1972
Titusville α94 (G1) Asp→ Asn Schneider et αl, 1975a Titusville α94 (G1) Asp → Asn Schneider and αl, 1975a
Mutants de la chaîne β Β chain mutants
Hémoglobine mutation Références Hemoglobin mutation References
Raleigh β1(NA1) Val→ac-Ala Moo-Penn et αl, 1977 Raleigh β1 (NA1) Val → ac-Ala Moo-Penn and αl, 1977
Connecricut β21 (B3) Asp→ Gly Moo-Penn et αl, 1981 Connecricut β21 (B3) Asp → Gly Moo-Penn and αl, 1981
Moscva β24 (B6) Gly→ Asp Idelson et αl, 1974 Moscva β24 (B6) Gly → Asp Idelson and αl, 1974
Auckland β25 (B7) Gly→ Asp Williamson et αl, 1987 Auckland β25 (B7) Gly → Asp Williamson and αl, 1987
Knossos β27 (B9) Ala→ Ser Baklouti et αl, 1986; Fessas et αl, 1982 Knossos β27 (B9) Ala → Ser Baklouti and αl, 1986; Fessas and αl, 1982
Rothschild β37 (C3) Trp→ Arg Gacon et αl, 1977 Rothschild β37 (C3) Trp → Arg Gacon et αl, 1977
Hazebrouck β38 (C4) Thr→Pro Blouquit et αl, 1984 Hazebrouck β38 (C4) Thr → Pro Blouquit et αl, 1984
Hammersmith β42 (CD1) Phe→ Ser Dacie et αl, 1967; Ohba et αl, 1975b Hammersmith β42 (CD1) Phe → Ser Dacie and αl, 1967; Ohba and αl, 1975b
Bucuresti β42 (CD1) Phe→Leu Bratu et αl, 1971; Keeling et αl, 1971 Tableau 1 (suite) Bucuresti β42 (CD1) Phe → Leu Bratu and αl, 1971; Keeling and αl, 1971 Table 1 (continued)
Sendagi β42 (CD1) Phe→Nal Ogata et αl, 1986 Sendagi β42 (CD1) Phe → Nal Ogata and αl, 1986
Chervely β45 (CD4) Phe→ Ser Yeager et αl, 1983 Chervely β45 (CD4) Phe → Ser Yeager et αl, 1983
Okaloosa β48 (CD7) Leu→ Arg Charache et αl, 1973 Okaloosa β48 (CD7) Leu → Arg Charache and αl, 1973
Bologna β61 (E5) Lys→ Met Marinucci et αl, 1981 Bologna β61 (E5) Lys → Met Marinucci and αl, 1981
J-Cairo β65 (E9) Lys→ Gin Gaiél et αl, 1976 J-Cairo β65 (E9) Lys → Gin Gaiél and αl, 1976
Chico β66 (E10) Lys→Thr Shih et αl, 1987 Chico β66 (E10) Lys → Thr Shih and αl, 1987
Bristol β67 (Ell) Val→Asp Steadman et αl, 1970 Bristol β67 (Ell) Val → Asp Steadman and αl, 1970
Seatde β70 (E14) Ala→ Asp Kurachi et α/, 1973 Seatde β70 (E14) Ala → Asp Kurachi and α /, 1973
Vancouver β73 (JEU) Asp→ Tyr Jones et αl, 1976 Vancouver β73 (GAME) Asp → Tyr Jones and αl, 1976
Korle-Bu β73 (E17) Asp→ Asn Konotey-Ahulu et αl, 1968; Korle-Bu β73 (E17) Asp → Asn Konotey-Ahulu and αl, 1968;
Wajcman & Jones, 1977 Wajcman & Jones, 1977
Mobile β73 (E17) Asp→ Val Schneider et αl, 1975b Mobile β73 (E17) Asp → Val Schneider and αl, 1975b
Tilburg β73 (E17) Asp→ Gly Bernini & Giordano, 1988 Tilburg β73 (E17) Asp → Gly Bernini & Giordano, 1988
Aalborg β74 (E18) Gly→Arg Williamson et αl, 1990 Aalborg β74 (E18) Gly → Arg Williamson and αl, 1990
Calais β76 (E20) Ala→Pro Wajcman et αl, 1991 Calais β76 (E20) Ala → Pro Wajcman and αl, 1991
Providence β82 (EF6) Lys→ Asn→ Asp Bonaventura ét αl, Moo-Penn et αl, 1976 Providence β82 (EF6) Lys → Asn → Asp Bonaventura et αl, Moo-Penn and αl, 1976
Pyrgos β83 (EF7) Gly→ Asp Taxsis et αl, 1976 Pyrgos β83 (EF7) Gly → Asp Taxsis et αl, 1976
Agenogi β90 (F6) Glu→ Lys Miyaji et αl, 1966 Agenogi β90 (F6) Glu → Lys Miyaji and αl, 1966
RoseauPoint-.e à Pitre β90 (F6) Glu→ Gly Mérault et αl, 1985 RoseauPoint-.e à Pitre β90 (F6) Glu → Gly Mérault et αl, 1985
Caribbean β91 (F7) Leu→ Arg Ahem et αl, 1976 Caribbean β91 (F7) Leu → Arg Ahem and αl, 1976
Kansas β102 (G4) Asn→Thr Bonaventura & Riggs, 1968 Kansas β102 (G4) Asn → Thr Bonaventura & Riggs, 1968
Beth Israel β102 (G4) Asn→Ser Νagel et αl, 1976 Beth Israel β102 (G4) Asn → Ser Νagel and αl, 1976
Saint- Mandé β102 (G4) Asn→Tyr Aiious et αl, 1981 Saint- Mandé β102 (G4) Asn → Tyr Aiious and αl, 1981
Burke β107 (G9) Giy→Arg Tumer et αl, 1976 Burke β107 (G9) Giy → Arg Tumer and αl, 1976
Yoshizuka β108 (G10) Asn→Asp Imamura et αl, 1969 Yoshizuka β108 (G10) Asn → Asp Imamura and αl, 1969
Presbyterian β108 (G10) Asn→Lys Moo-Penn et αl, 1978 Tableau 1 (suite) Presbyterian β108 (G10) Asn → Lys Moo-Penn and αl, 1978 Table 1 (continued)
Peterborough β111(G13)Val→Phe King et αl, 1972 Peterborough β111 (G13) Val → Phe King and αl, 1972
New-York β113(G15)Val→Glu Ranney et αl, 1967 New-York β113 (G15) Val → Glu Ranney and αl, 1967
D-Punjab β121(GH4)Glu→Gln Baglioni, 1962 D-Punjab β121 (GH4) Glu → Gln Baglioni, 1962
La Desirade β129(H7)Ala→Val Mérault et αl, 1986 La Desirade β129 (H7) Ala → Val Mérault and αl, 1986
Yamagata β132(H10)Lys→Asn Harano et αl, 1990Yamagata β132 (H10) Lys → Asn Harano and αl, 1990
Hope β136(H14)Gly→Asp Minnich et αl, 1965Hope β136 (H14) Gly → Asp Minnich and αl, 1965
Himeji β140(H18)Ala→Asp Ohba et αl, 1986 Himeji β140 (H18) Ala → Asp Ohba and αl, 1986
NB : n'ont été pris en compte que les hémoglobines contenant une seule mutation soit dans la sousunité α , soit dans la sous-unité β. NB: only hemoglobins containing a single mutation were taken into account either in the α subunit or in the β subunit.
Comme cela a été indiqué précédemment, les séquences W et Y doivent permettre de reconstituer une interface alpha 1 bêta2 de HbA ou similaire qui est impliquée dans la transition R—> T. C'est pourquoi, les segments (W) et (Y) seront similaires aux segments C/CD et FG/G de l'interface alpha, bêta2 de l'hémoglobine humaine, mais il est possible de la modifier si besoin est en prévoyant certaines mutations ponctuelles. As indicated above, the W and Y sequences must make it possible to reconstitute an alpha 1 beta 2 interface for HbA or the like which is involved in the transition R—> T. This is why the segments (W) and (Y ) will be similar to the C / CD and FG / G segments of the alpha, beta 2 interface of human hemoglobin, but it is possible to modify it if necessary by predicting certain point mutations.
Les éléments essentiels impliqués dans la structure de l'interface alpha 1 bêta2 sont les suivants : The essential elements involved in the structure of the alpha 1 beta 2 interface are as follows:
Chaîne Chaîne Chaîne Chaîne Chain Chain Chain Chain
Bêta Alpha Bêta Alpha Beta Alpha Beta Alpha
37Trp 38Thr Tyr 145 Tyr 140 37Trp 38Thr Tyr 145 Tyr 140
40Arg 41Thr Asn 102 Asn 97 40Arg 41Thr Asn 102 Asn 97
41 Phe 42Tyr Glu 101 Val 96 41 Phe 42Tyr Glu 101 Val 96
96Leu 91 Leu Asp 99 Asp 94 96Leu 91 Leu Asp 99 Asp 94
97His 92Arg His 97 Arg 92 97His 92Arg His 97 Arg 92
98Vai 93Val Phe 41 Tyr 42 98Vai 93Val Phe 41 Tyr 42
99 Asp 94Asp Arg 40 Thr 41 99 Asp 94Asp Arg 40 Thr 41
100Pro 95Pro Gln 39 Lys 40 100Pro 95Pro Gln 39 Lys 40
101Glu 96 Val Trp 37 Thr 38 101Glu 96 Val Trp 37 Thr 38
145Tyr 140Tyr Pro 36 Pro 37 145Tyr 140Tyr Pro 36 Pro 37
On peut prévoir les mutations figurant au tableau 2 ci-après. We can predict the mutations in Table 2 below.
Exemplesde mutations de résidus situés à l'interface α1β2 Examples of mutations of residues located at the α1β2 interface
Site mutation particularité de la mutation- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - β40 (C6) Arg→ Asp charge négative Mutation site particularity of the mutation - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - β40 (C6) Arg → Asp negative charge
encombrement plus faible que Arg Arg-→ Val hydrophobe smaller footprint than Arg Arg- → Hydrophobic Val
fort encombrement stérique Arg→ Ala hydrophobe strong steric hindrance Arg → Hydrophobic Ala
faible encombrement stérique Arg→ Ser groupement hydroxyle small steric hindrance Arg → Ser hydroxyl group
faible encombrement stérique Arg→ Lys propriétés proches de celle de Arg β41 (C7) Phe→ Tyr groupement hydroxyle small steric hindrance Arg → Lys properties close to that of Arg β41 (C7) Phe → Tyr hydroxyl group
possibilité de liaison H β101 (G3) Glu→Ala hydrophobe possibility of H β101 (G3) Glu → Ala hydrophobic bonding
faible encombrement stérique β102 (G4) Asn→ Ala hydrophobe small steric hindrance β102 (G4) Asn → Hydrophobic Ala
faible encombrement stérique Asn→ Gly encombrement stérique nul- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - -- - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - low steric hindrance Asn → Gly steric hindrance zero - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Parmi les chaînes polypeptidiques plus particulièrement intéressantes, il faut citer les chaînes mixtes alpha,bêta, c'est-à-dire notamment les chaînes constituées de l'extrémité N-terminale d'une chaîne de type bêta de la globine, et de l'extrémité C-terminale de la chaîne alpha de la globine ; ou, réciproquement, une chaîne constituée de l'extrémité N-terminale d'une chaîne alpha de la globine et de l'extrémité C-terminale d'une chaîne de type bêta de la globine. Among the polypeptide chains which are more particularly advantageous, mention should be made of the mixed alpha, beta chains, that is to say in particular the chains consisting of the N-terminal end of a beta-type chain of globin, and of the C-terminus of the globin alpha chain; or, conversely, a chain consisting of the N-terminal end of an alpha chain of globin and the C-terminal end of a beta-type chain of globin.
Ces sous-unités chimériques seront notées, respectivement, NH2bêta-aiphaCOOH ou NH2alpha-bêtaCOOH. These chimeric subunits will be noted, respectively, NH 2 beta-aiphaCOOH or NH 2 alpha-betaCOOH.
La jonction alpha-bêta se situera de préférence au niveau de la zone flexible de la poche de Thème, c'est-à-dire dans le segment EF. The alpha-beta junction will preferably be located in the flexible area of the Theme pocket, that is to say in the EF segment.
Le polypeptide le plus intéressant comporte l'extrémité N-terminale de la chaîne bêta de la globine fusionnée jusqu'à Tamino-acide 73 compris à la partie C-terminale de la chaîne alpha de la globine à partir de Tamino-acide 69 compris. The most interesting polypeptide comprises the N-terminal end of the beta chain of globin fused to Tamino-acid 73 included in the C-terminal part of the alpha chain of globin from Tamino-acid 69 included.
Ce type de molécule chimérique associée sous forme tétramérique est représenté à la figure 8 sur laquelle on a visualisé les interfaces alpha 1 bêta2 reconstituées dans le tétramère. This type of chimeric molecule associated in tetrameric form is represented in FIG. 8 in which the alpha 1 beta 2 interfaces reconstituted in the tetramer have been visualized.
Il est possible de prévoir que deux chaînes polypeptidiques soient reliées par une séquence, de préférence peptidique, située entre la séquence Z de Tune et la séquence V de l'autre, les deux chaînes sont ainsi solidaires, ce qui favorise la stabilité du tétramère. It is possible to provide for two polypeptide chains to be linked by a sequence, preferably peptide, located between the Z sequence of Tune and the V sequence of the other, the two chains are thus integral, which promotes the stability of the tetramer.
Les deux chaînes peuvent également être reliées par reticulation ou par tout type d'interaction biochimique comme par exemple des liaisons de pontage covalent (dérivés de diaspirine ou de pyridoxal phosphate). The two chains can also be linked by crosslinking or by any type of biochemical interaction such as, for example, covalent bridging bonds (derivatives of diaspirin or of pyridoxal phosphate).
Le transporteur d'oxygène selon l'invention comportera une structure héminique ; il peut s'agir d'une molécule d'hème naturel ou bien d'une métalioporphyrine dans laquelle l'atome de fer est remplacé par un autre métal notamment le cobalt. Dans le second mode de réalisation de l'invention, le tétramère est constitué essentiellement de quatre polypeptides identiques ayant chacun une structure de type bêta-globine. Ce type de tétramère dénommé bêta4 sera, de préférence, constitué par des chaînes de type bêta comportant Tune des mutations citées précédemment. The oxygen transporter according to the invention will include a heme structure; it may be a natural heme molecule or a metalioporphyrin in which the iron atom is replaced by another metal, in particular cobalt. In the second embodiment of the invention, the tetramer consists essentially of four identical polypeptides each having a beta-globin type structure. This type of tetramer called beta 4 will preferably be constituted by beta type chains comprising one of the mutations mentioned above.
Il pourra, comme cela a été décrit, être préparé par association de dimères, ces dimères étant formés par liaison des extrémités N et C terminales de deux sous-unités. It could, as has been described, be prepared by association of dimers, these dimers being formed by bonding the N and C ends of two subunits.
Le transporteur d'oxygène selon l'invention présente en général une affinité pour l'oxygène diminuée par rapport à l'hémoglobine naturelle, de préférence, cette affinité est diminuée d'au moins 20 % par rapport à celle de l'hémoglobine naturelle. The oxygen transporter according to the invention generally has a reduced affinity for oxygen compared to natural hemoglobin, preferably this affinity is reduced by at least 20% compared to that of natural hemoglobin.
Le transporteur selon la présente invention est, de préférence, obtenu par culture d'un organisme transformé produisant ladite chaîne, il peut s'agir de cellules eucaryotes comportant une séquence d'ADN codant pour ladite chaîne sous la dépendance d'éléments d'expression assurant l'expression de ladite séquence dans ladite cellule hôte. The transporter according to the present invention is preferably obtained by culture of a transformed organism producing said chain, it may be eukaryotic cells comprising a DNA sequence coding for said chain under the dependence of expression elements ensuring the expression of said sequence in said host cell.
La technologie des ADN recombinants permettant l'expression d'une séquence peptidique est maintenant largement connue et n'a pas à être décrite en détail. The technology of recombinant DNA allowing the expression of a peptide sequence is now widely known and need not be described in detail.
La cellule hôte est de préférence une cellule eucaryote et de préférence une levure et/ou une cellule eucaryote supérieure ou un animal transgénique. Quant au système d'expression, il peut s'agir soit de plasmides à réplication autonome, soit de piasmides permettant Tintégratioή dans le génome de la cellule hôte. Les séquences codantes pouvant être sous la dépendance d'un promoteur hétérologue mais efficace dans l'hôte ou bien d'un promoteur homologue, ou bien encore d'un promoteur chromosomique. Il est possible également de prévoir des systèmes assurant, par exemple, la sécrétion de ces chaînes polypeptidiques. The host cell is preferably a eukaryotic cell and preferably a yeast and / or a higher eukaryotic cell or a transgenic animal. As for the expression system, it may be either plasmids with autonomous replication, or piasmids allowing integration into the genome of the host cell. The coding sequences may be dependent on a heterologous but effective promoter in the host or on a homologous promoter, or even on a chromosomal promoter. It is also possible to provide systems ensuring, for example, the secretion of these polypeptide chains.
Dans ce qui suit, on donnera des exemples de système d'expression des chaînes polypeptidiques selon la présente invention qui ne pourront, en aucun cas, être considérées comme des limitations. In the following, examples of the expression system of the polypeptide chains according to the present invention will be given which cannot, in any case, be considered as limitations.
L'obtention des tétramères est, la plupart du temps, spontanée, elle peut être favorisée suivant la structure exacte du tétramère par des conditions particulières de mise en solution, notamment dans certaines conditions de pH et/ou de force ionique. Obtaining tetramers is, most of the time, spontaneous, it can be favored according to the exact structure of the tetramer. by special conditions for dissolving, in particular under certain pH and / or ionic strength conditions.
L'introduction d'un groupe héminique peut être spontanée, en particulier chez les eucaryotes, et les tétramères obtenus sont "héminisés" sans qu'il y ait lieu de prévoir une étape spécifique. La plupart du temps Thème sera rajouté lors d'une étape ultérieure. The introduction of a hemin group can be spontaneous, in particular in eukaryotes, and the tetramers obtained are "heminized" without there being any need for a specific step. Most of the time Theme will be added at a later stage.
La présente invention concerne également des procédés permettant la préparation de ces chaînes polypeptidiques par des techniques mettant en oeuvre les ADN recombinants et l'éventuelle formation du tétramère, comme cela a été décrit précédemment (Hoffman et αl, 1990 ; Nagai & Thogersen, 1984, 1987 ; Nagai et αl, 1985 ; Tame et αl, 1991 ; Wagenbach et αl, 1991). The present invention also relates to methods allowing the preparation of these polypeptide chains by techniques using the recombinant DNAs and the possible formation of the tetramer, as described above (Hoffman and αl, 1990; Nagai & Thogersen, 1984, 1987; Nagai and αl, 1985; Tame and αl, 1991; Wagenbach and αl, 1991).
Enfin, la présente invention concerne l'utilisation des chaînes à titre de médicament, notamment pour le transport d'oxygène en combinaison avec un support acceptable dans le domaine transfusionnel. Finally, the present invention relates to the use of chains as a medicament, in particular for the transport of oxygen in combination with an acceptable support in the transfusion field.
Les exemples ci-après permettront de mieux mettre en évidence certaines caractéristiques et avantages de la présente invention. The following examples will better illustrate certain characteristics and advantages of the present invention.
Dans ce qui suit : In the following :
- la Figure 1 schématise la séquence nuciéotidique et en amino-acides de la chaîne alpha, - Figure 1 shows schematically the nucleotide and amino acid sequence of the alpha chain,
- la Figure 2 schématise la séquence nuciéotidique et en amino-acides de la chaîne bêta, FIG. 2 schematizes the nucleotide and amino acid sequence of the beta chain,
- la Figure 3 schématise la structure spatiale de la chaîne bêta, avec indication de la nomenclature des hélices, - Figure 3 shows schematically the spatial structure of the beta chain, with indication of the nomenclature of the propellers,
- la Figure 4 représente le schéma du plasmide pATPrTet, FIG. 4 represents the diagram of the plasmid pATPrTet,
- la Figure 5 représente le schéma du plasmide pATPr cIIFX bêta, FIG. 5 represents the diagram of the plasmid pATPr cIIFX beta,
- les Figures 6 et 7 schématisent la préparation du vecteur pATPr cIIFX bêta Gb, FIGS. 6 and 7 show diagrammatically the preparation of the vector pATPr cIIFX beta Gb,
- la Figure 8 représente le schéma de la structure spatiale d'un dimère selon l'invention, notamment la reconstitution de l'interface alpha 1 bêta2. FIG. 8 represents the diagram of the spatial structure of a dimer according to the invention, in particular the reconstruction of the alpha 1 beta 2 interface.
- la Figure 9 schématise les étapes de la construction du vecteur pATPr Chim bêta73-alpha69. Afin de mieux comprendre la structure de l'hémoglobine, on pourra se reporter à des articles tels que celui de Max F. Perutz ( 1987) in Molecular Basis of Blood Diseases ainsi qu'à des ouvrages tels ceux de Dickerson & Geis (1983) et Fermi & Perutz ( 1981). - Figure 9 schematizes the stages of the construction of the vector pATPr Chim beta73-alpha69. In order to better understand the structure of hemoglobin, reference may be made to articles such as that of Max F. Perutz (1987) in Molecular Basis of Blood Diseases as well as to works such as those of Dickerson & Geis (1983) & Fermi & Perutz (1981).
Sur la figure 1 on a représenté la structure en amino-acides de la chaîne alpha et sur la figure 2 la structure en amino-acides de la chaîne bêta. In Figure 1 the amino acid structure of the alpha chain is shown and in Figure 2 the amino acid structure of the beta chain.
La configuration spatiale de la chaîne bêta est représentée à la figure 3 avec la nomenclature des différents segments qui vont de l'extrémité NH2 terminale vers l'extrémité COOH terminale : The spatial configuration of the beta chain is represented in FIG. 3 with the nomenclature of the various segments which go from the NH 2 terminal end towards the COOH terminal end:
NA, A, B, C, CD, D, E, EF, F, FG, G, GH, H et HC ; NA, A, B, C, CD, D, E, EF, F, FG, G, GH, H and HC;
quant à la chaîne alpha elle présente la structure : as for the alpha chain it has the structure:
NA, A, AB, B, C, CE, E, EF, F, FG, G, GH, H et HC. NA, A, AB, B, C, CE, E, EF, F, FG, G, GH, H and HC.
Les segments A, B, C, E, F, G et H présentent une structure sensiblement hélicoïdale alors que les segments de jonction ne présentent pas une telle structure. The segments A, B, C, E, F, G and H have a substantially helical structure while the junction segments do not have such a structure.
Les acides aminés sont numérotés soit à partir de l'extrémité N-terminale par ordre croissant, soit en affectant à un acide aminé la désignation de son hélice et son rang dans celle-ci. Amino acids are numbered either from the N-terminal end in ascending order, or by assigning an amino acid the designation of its helix and its rank in it.
Le 36ème acide aminé de la chaîne bêta est la Proline, il est bêta 36(C2) Pro, c'est-à-dire le deuxième acide aminé de Thélice C. The 36th amino acid in the beta chain is Proline, it is beta 36 (C2) Pro, i.e. the second amino acid in Thélice C.
PRODUCTION DE LA PROTEINE DE FUSION DANS E. COLI Les manipulations portant sur l' ADN ont été effectuées en utilisant essentiellement les techniques classiques décrites par Sambrook et al dans le manuel "Molecular Cloning". Sauf indication contraire, les digestions par les enzymes de restriction ou de modification sont réalisées selon les indications des fabricants (Amersham, Boehringer Manheim; New England Biolabs). L' ADNc codant pour la protéine de fusion cIIFX bêta globine a été clone par Nagai et al (1985) dans le phage M13 mp 10. La construction M13 mp 10 cIIFX bêta nous a été fournie sur demande au Médical Research Council (Cambridge, Grande-Bretagne). EXEMPLE 1 : Construction du vecteur d'expression p AT Pr CIIFX bêta Gb PRODUCTION OF THE FUSION PROTEIN IN E. COLI The manipulations relating to DNA were carried out essentially using the standard techniques described by Sambrook et al in the manual "Molecular Cloning". Unless otherwise indicated, digestions with restriction or modification enzymes are carried out according to the manufacturers' indications (Amersham, Boehringer Manheim; New England Biolabs). The cDNA coding for the fusion protein cIIFX beta globin was cloned by Nagai et al (1985) into the phage M13 mp 10. The construct M13 mp 10 cIIFX beta was supplied to us on request to the Medical Research Council (Cambridge, Grande -Brittany). EXAMPLE 1 Construction of the expression vector p AT Pr CIIFX beta Gb
Le vecteur de base pAT Pr Tet présente une origine de répiication dérivée de pBR 322 et porte le gène de résistance à la tétracycline. Il permet l'expression de protéines hétérologues sous le contrôle du promoteur Pr du phage lambda. L'activité de ce promoteur est réprimée par le répresseur CI (ailèle CI8 57, thermosensible). Le promoteur Pr est bloqué à 30° C. L'élévation de la température à 42° C inactive le répresseur et induit la transcription à partir du promoteur Pr (figure 4). The basic vector pAT Pr Tet has an origin of replication derived from pBR 322 and carries the gene for resistance to tetracycline. It allows the expression of heterologous proteins under the control of the Pr promoter of phage lambda. The activity of this promoter is repressed by the repressor CI (ailèle CI 8 57 , thermosensitive). The Pr promoter is blocked at 30 ° C. Raising the temperature to 42 ° C inactivates the repressor and induces transcription from the Pr promoter (FIG. 4).
Préparation de l' ADN plasmidique Preparation of plasmid DNA
Les bactéries E. coli CAG 1139 portant le plasmide sont mises en culture à 37° C (1 L milieu LB) jusqu'à obtention d'une DO à 600 nm d'environ 0,4. On ajoute du chloramphénicol à une concentration finale de 170 μg/ml, et on continue l'incubation à 37° C pendant 12-16 heures. Les bactéries sont lysées par traitement avec une solution de NaOH-SDS, et TADN plasmidique est purifié à partir du culot bactérien par précipitation avec le polyéthylène giycol. Le protocole utilisé est très exactement celui décrit par Sambrook et al (1989). The E. coli CAG 1139 bacteria carrying the plasmid are cultured at 37 ° C. (1 L LB medium) until an OD at 600 nm of approximately 0.4 is obtained. Chloramphenicol is added to a final concentration of 170 μg / ml, and the incubation is continued at 37 ° C for 12-16 hours. The bacteria are lysed by treatment with a NaOH-SDS solution, and the plasmid DNA is purified from the bacterial pellet by precipitation with polyethylene giycol. The protocol used is very exactly that described by Sambrook et al (1989).
10 μg d' ADN plasmidique pAT Pr Tet ainsi purifié sont incubés en présence de 5 unités d'endonuciéase de restriction BamHl, 10 minutes à 37° C (digestion ménagée). La forme linéaire du plasmide est séparée sur gel d'agarose 0,8 %, et recueillie par électroélution. L'ADN est précipité par Téthanol après extraction au phénol/chloroforme. Le précipité est repris dans 90 μl d'eau distillée et traité par la nuclease de haricot mung (mung bean nuclease, New England Biolabs) de façon à obtenir le codon d'initiation ATG sous forme d'extrémité franche. Après extraction au phénol/chloroforme et précipitation par Téthanol, l' ADN est dissout dans 90 μl d'eau et incubé en présence de 100 unités d'endonuciéase de restriction Xhol (figure 6). Le fragment BamHI/Xho de 4500 bp est séparé par électrophorèse sur gel d'agarose, recueilli par électroélution, et précipité par Téthanol après extraction au phénol/chloroforme. Préparation de la séquence à insérer 10 μg of plasmid DNA pAT Pr Tet thus purified are incubated in the presence of 5 units of restriction endonuciasis BamHI, 10 minutes at 37 ° C (digestion managed). The linear form of the plasmid is separated on 0.8% agarose gel, and collected by electroelution. The DNA is precipitated with tethanol after extraction with phenol / chloroform. The precipitate is taken up in 90 μl of distilled water and treated with mung bean nuclease (mung bean nuclease, New England Biolabs) so as to obtain the initiation codon ATG in the form of a blunt end. After phenol / chloroform extraction and precipitation with tethanol, the DNA is dissolved in 90 μl of water and incubated in the presence of 100 units of Xhol restriction endonuciasis (FIG. 6). The BamHI / Xho fragment of 4500 bp is separated by agarose gel electrophoresis, collected by electroelution, and precipitated with tethanol after extraction with phenol / chloroform. Preparation of the sequence to be inserted
La séquence codant pour la protéine de fusion a été obtenue par la technique de PCR à partir de la forme réplicative double brin du phage M 13 mp10 (10 ng). La forme réplicative double brin est préparée selon la technique indiquée ci-dessus pour l' ADN plasmidique, à partir d'un culot bactérien E. coli, souche JM 101. Un site reconnu par Tendonuclease de restriction SnaBI est introduit à l'extrémité 5' de la séquence codante.The sequence coding for the fusion protein was obtained by the PCR technique from the double-stranded replicative form of phage M 13 mp10 (10 ng). The double-stranded replicative form is prepared according to the technique indicated above for the plasmid DNA, from an E. coli bacterial pellet, strain JM 101. A site recognized by SnaBI restriction tendonuclease is introduced at the end 5 'of the coding sequence.
La coupure par cette enzyme génère une extrémité franche correspondant au premier nucléotide du premier codon de la protéine de fusion. Un site reconnu par Tendonuclease de restriction Xhol est créé à l'extrémité 3', immédiatement après le codon stop (Fig. 7). Le produit de PCR est précipité par Téthanol après extraction au phénol/chloroforme. Le précipité est repris par 90 μl de tampon Tns-HCl 10 mM/ EDTA 1 mM. L'ADN est traité par les endonucléases de restriction SnaBI (15 unités, 2 heures, 37°Cleavage by this enzyme generates a blunt end corresponding to the first nucleotide of the first codon of the fusion protein. A site recognized by Tendonuclease of Xhol restriction is created at the 3 'end, immediately after the stop codon (FIG. 7). The PCR product is precipitated with tethanol after extraction with phenol / chloroform. The precipitate is taken up in 90 μl of 10 mM Tns-HCl / 1 mM EDTA buffer. DNA is treated with SnaBI restriction endonucleases (15 units, 2 hours, 37 °
C) puis Xhol (25 unités, 2 heures, 37° C). Il est précipité par Téthanol après extraction au phénol/chloroforme. Le précipité est repris dans 10 μl de tampon Tris-HCl 10 mM/EDTA 1 mM. L'insertion dans le vecteur d'expression est réalisée en utilisant un rapport vecteur/insert de 1/5 (soit 3 μl de la solution d'ADN vecteur et 3 μl de la solution d'insert). Le mélange est incubé en présence de T4 ADN ligase, 4 heures à température ambiante. C) then Xhol (25 units, 2 hours, 37 ° C). It is precipitated with tethanol after extraction with phenol / chloroform. The precipitate is taken up in 10 μl of 10 mM Tris-HCl / 1 mM EDTA buffer. The insertion into the expression vector is carried out using a vector / insert ratio of 1/5 (i.e. 3 μl of the vector DNA solution and 3 μl of the insert solution). The mixture is incubated in the presence of T4 DNA ligase, 4 hours at room temperature.
Le mélange réactionnel est utilisé pour transformer des bactéries CAG 1 139 compétentes (traitement CaCl2). Les bactéries transformées sélectionnées par la résistance à la tetracycline sont testées pour l'expression de la protéine de fusion. The reaction mixture is used to transform competent CAG 1139 bacteria (CaCl 2 treatment). The transformed bacteria selected by the resistance to tetracycline are tested for the expression of the fusion protein.
Le vecteur ainsi obtenu pAT Pr cIIFX bêta dirige la synthèse d'une protéine de fusion cIIFX bêta globine contenant : The vector thus obtained pAT Pr cIIFX beta directs the synthesis of a fusion protein cIIFX beta globin containing:
- les 31 résidus N-terrrunaux de la protéine CII du phage lambda ; - le tétrapeptide Ile-Glu-Gly-Arg, séquence reconnue par le facteur de coagulation X activé, ce qui permet de libérer la sous-unité bêta globine après purification de la protéine de fusion ; - the 31 N-terrrunal residues of the protein CII of the phage lambda; - the Ile-Glu-Gly-Arg tetrapeptide, sequence recognized by activated coagulation factor X, which makes it possible to release the beta globin subunit after purification of the fusion protein;
- la bêta globine. EXEMPLE 2 : Expression de la protéine dans Escherichia coli - beta globin. EXAMPLE 2 Expression of the protein in Escherichia coli
La souche E. coli CAG 1139 portant le plasmide pAT Pr cIIFX bêta est incubée à 30° C dans du milieu M9 (Na2HPO 4 50 mM/KH 2PO 420 mM/NaCI 8,5 mM/NH4Cl20mM/MgSO^ O.lmM) contenant 2 g de glucose, 20g d'extrait de levure et 0.5 g de vitamine B1 par litre) en présence de tetracycline (10 mg par litre). Lorsque Tabsorbance à 600 nm atteint 0,8-1,2, on élève rapidement la température à 42° C, et Ton continue l'incubation pendant 3-4 heures. Le culot bactérien est congelé à -80° C. The E. coli CAG 1139 strain carrying the plasmid pAT Pr cIIFX beta is incubated at 30 ° C in M9 medium (Na 2 HPO 4 50 mM / KH 2 PO 4 20 mM / NaCI 8.5 mM / NH 4 Cl20 mM / MgSO ^ O.lmM) containing 2 g of glucose, 20 g of yeast extract and 0.5 g of vitamin B1 per liter) in the presence of tetracycline (10 mg per liter). When the absorbance at 600 nm reaches 0.8-1.2, the temperature is rapidly raised to 42 ° C., and the incubation is continued for 3-4 hours. The bacterial pellet is frozen at -80 ° C.
EXEMPLE 3 : Extraction et purification de la protéine de fusion EXAMPLE 3 Extraction and purification of the fusion protein
Le culot bactérien ( 100 g poids humide) est décongelé et mis en suspension dans 100 ml Tris-HCl 50 mM, pH 8/ saccharose 25 % (p/v)/EDTA 1 mM. Les bactéries sont lysées par incubation en présence de 200 mg de lysozyme (30 minutes dans la glace). On ajoute du MgCl2, du MnCl2 et de la DNase I de façon à obtenir des concentrations finales respectives de 10 mM, 1 mM et 10 μg/ml. Après 30 min d'incubation à température ambiante, on ajoute au lysat 200 ml de NaCl 0,2M/ acide désoxycholique 1 % (p/v)/Nonιdet P-40 1,6 % (v/v)/Tris-HCl 20 mM, pH 7,5/EDTA 2 mM. Le précipité est recueilli par centrifugation à 5000 g, 10 mm, remis en suspension dans du Triton X-100 1 % (v/v)/EDTA 1 mM et centrifugé à nouveau. Les lavages dans la solution de Triton X-100 sont renouvelés jusqu'à obtention d'un précipité compact. The bacterial pellet (100 g wet weight) is thawed and suspended in 100 ml 50 mM Tris-HCl, pH 8/25% sucrose (w / v) / 1 mM EDTA. The bacteria are lysed by incubation in the presence of 200 mg of lysozyme (30 minutes in ice). MgCl 2 , MnCl 2 and DNase I are added so as to obtain respective final concentrations of 10 mM, 1 mM and 10 μg / ml. After 30 min of incubation at room temperature, 200 ml of 0.2M NaCl / 1% deoxycholic acid (w / v) / Nonιdet P-40 1.6% (v / v) / Tris-HCl 20 are added to the lysate. mM, pH 7.5 / 2 mM EDTA. The precipitate is collected by centrifugation at 5000 g, 10 mm, resuspended in 1% Triton X-100 (v / v) / 1 mM EDTA and centrifuged again. The washes in the Triton X-100 solution are repeated until a compact precipitate is obtained.
Le précipité de protéine est dissout dans une solution de guanidine-HCl 6M/Tris-AcOH 25 mM, pH 5,0/EDTA 1 mM/dithiothreitol 1 mM. La solution est équilibrée dans un tampon urée 8M/Tris-AcOH 25 mM, pH 5,0/EDTA 1 mM/dithiothreitol 1 mM par filtration sur une colonne de Sephadex G-25, puis déposée sur une colonne de CM-Sepharose (4 × 10 cm) équilibrée avec le même tampon. La protéine de fusion est éiuée à l'aide d'un gradient linéaire formé par 500 ml d'urée 8M/Tris-AcOH 25 mM, pH 5,0/EDTA 1 mM/dithiothreitol 1 mM et 500 ml du même tampon additionné de NaCl qsp une concentration de 0,2 M. La protéine de fusion est ensui te purifiée par filtration sur colonne de Sephacryl S-200 (5 × 60 cm) en milieu guanidine-HCl 5 M/Tris-HCl 50 mM, pH 8,0/EDTA 1 mM/dithiothreitol 1 mM. Les fractions contenant la protéine sont rassemblées, dialysées contre de l'eau et lyophilisées, ou dialysées contre le tampon de clivage pour être traitées immédiatement par le facteur X activé. The protein precipitate is dissolved in a solution of guanidine-HCl 6M / Tris-AcOH 25 mM, pH 5.0 / EDTA 1 mM / dithiothreitol 1 mM. The solution is equilibrated in an 8M urea buffer / 25 mM Tris-AcOH, pH 5.0 / 1 mM EDTA / 1 mM dithiothreitol by filtration through a column of Sephadex G-25, then deposited on a column of CM-Sepharose (4 × 10 cm) balanced with the same buffer. The fusion protein is eluted using a linear gradient formed by 500 ml of 8M urea / 25 mM Tris-AcOH, pH 5.0 / EDTA 1 mM / dithiothreitol 1 mM and 500 ml of the same buffer supplemented with NaCl qsp a concentration of 0.2 M. The fusion protein is then purified by filtration on a column of Sephacryl S-200 (5 × 60 cm) in guanidine-5 M HCl / 50 mM Tris-HCl, pH 8.0 / 1 mM EDTA / 1 mM dithiothreitol. The protein-containing fractions are pooled, dialyzed against water and lyophilized, or dialyzed against the cleavage buffer to be immediately treated with activated factor X.
Après purification de la protéine de fusion et hydrolyse trypsique, la présence de la mutation et le profil peptidique sont vérifiés par chromatographie liquide haute performance (Baudin-Chich et al, 1987). After purification of the fusion protein and tryptic hydrolysis, the presence of the mutation and the peptide profile are verified by high performance liquid chromatography (Baudin-Chich et al, 1987).
EXEMPLE 4 : Clivage de la protéine de fusion par le facteur Xa La protéine lyophilisée est dissoute dans une solution d'uréeEXAMPLE 4 Cleavage of the fusion protein by factor Xa The lyophilized protein is dissolved in a urea solution
8M/Tris-HCi 50 mM, pH 8.0/EDTA 1 mM/dithiothreitol 1 mM, puis dialysée contre le tampon de clivage : urée 0,5M/Tris-HCl 50mM, pH 8,0/CaCl2 1 mM. La protéine de fusion est incubée à 0° C en présence de facteur X activé par le venin de vipère de Russel immobilisé sur Sepharose 6B activé au bromure de cyanogène (rapport enzyme/substrat 1 ,5 % p/p). La qualité du clivage est vérifiée par électrophorèse sur gel de polyacrylamide-SDS. Après dialyse contre de Teau, la protéine est lyophilisée. 8M / 50 mM Tris-HCl, pH 8.0 / 1 mM EDTA / 1 mM dithiothreitol, then dialyzed against the cleavage buffer: 0.5 M urea / 50 mM Tris-HCl, pH 8.0 / CaCl 2 1 mM. The fusion protein is incubated at 0 ° C. in the presence of factor X activated by Russell's viper venom immobilized on Sepharose 6B activated with cyanogen bromide (enzyme / substrate ratio 1.5% w / w). The quality of the cleavage is verified by electrophoresis on polyacrylamide-SDS gel. After dialysis against water, the protein is lyophilized.
EXEMPLE 5 : Construction du mutant bétaH2M EXAMPLE 5 Construction of the betaH2M mutant
La mutation est introduite dans TADN en utilisant un oligonuciéôtide initiateur synthétique de séquence dC AGG AGT CAG CAT CAC CCT ACC C. Cette séquence est complémentaire à celle de TADNc codant pour la sous-unité bêta de globine encadrant le codon de Thistidine bêta (NA2). Le triplet introduit CAT (au lieu de GTG) est altéré de telle sorte à coder pour une methionine. La mutagénèse est réalisée en utilisant la réplication du phage M 13 dans lequel est inséré TADNc codant pour la chaîne bêta de globine (Nagai & Thogersen, 1984), par la méthode de Nakamaye & Eckstein ( 19S6) préconisée par Amersham (Kit Amersham). Le produit de la réaction de mutagénèse est utilisé pour transformer des bactéries compétentes E. coli, souche JM 101 selon les méthodes classiques (Nagai & Thogersen, 1987 ; Sambrook et al, 1989). Les phages portant la mutation sont détectés par séquençage de T ADN (méthode de Sanger, 1977). La séquence complète de TADNc codant pour la protéine de fusion est vérifiée de façon à s'assurer de l'intégrité de la séquence codante. The mutation is introduced into TADN using a synthetic oligonucleotide initiator of sequence dC AGG AGT CAG CAT CAC CCT ACC C. This sequence is complementary to that of cDNA coding for the beta subunit of globin framing the codon of Thistidine beta (NA2) . The triplet introduced CAT (instead of GTG) is altered so as to code for a methionine. Mutagenesis is performed using the replication of the phage M 13 into which is inserted cDNA coding for the beta chain of globin (Nagai & Thogersen, 1984), by the method of Nakamaye & Eckstein (19S6) recommended by Amersham (Kit Amersham). The product of the mutagenesis reaction is used to transform competent E. coli bacteria, strain JM 101 according to conventional methods (Nagai & Thogersen, 1987; Sambrook et al, 1989). The phages carrying the mutation are detected by T DNA sequencing (Sanger method, 1977). The complete sequence of cDNA coding for the fusion protein is verified in order to ensure the integrity of the coding sequence.
La séquence codante contenant la mutation est traitée comme décrit ci-dessus pour être insérée dans le vecteur d'expression. The coding sequence containing the mutation is processed as described above to be inserted into the expression vector.
EXEMPLE 6 : Construction du mutant bêtaH21 EXAMPLE 6 Construction of the betaH21 mutant
La mutation est introduite dans TADNc en utilisant Toligonuciéotide initiateur dAGG AGT CAG GAT CAC CCT ACC C. Le triplet GAT (pour GTG) permet de coder pour une isoieucine. The mutation is introduced into cDNA using Toligonuciéotide initiator dAGG AGT CAG GAT CAC CCT ACC C. The triplet GAT (for GTG) makes it possible to code for an isoieucine.
EXEMPLE 7 : Construction du mutant bêtaFMY EXAMPLE 7 Construction of the betaFMY mutant
La mutation est introduite dans TADNc en utilisant Tohgonucléotide initiateur dGGA CTC AAA GTA CCT CTG GGT. Le triplet GTA (pour GAA) permet de coder pour une tyrosine. The mutation is introduced into cDNA using Tohgonucleotide initiator dGGA CTC AAA GTA CCT CTG GGT. The triplet GTA (for GAA) makes it possible to code for a tyrosine.
EXEMPLE 8 : Construction du vecteur pAT Pr Chim bêta73-alpha69 EXAMPLE 8 Construction of the vector pAT Pr Chim beta73-alpha69
La construction codant pour la chimère bêta-alpha globine est construite par PCR à partir de deux plasmides : The construction coding for the beta-alpha globin chimera is constructed by PCR from two plasmids:
- le plasmide pAT Pr bêta, portant le gène bêta globine (exemple 1 ), - the plasmid pAT Pr beta, carrying the beta globin gene (example 1),
- un plasmide portant TADNc alpha globine : alphal. - a plasmid carrying cDNA alpha globin: alphal.
On utilise les amorces suivantes : Nsi : 5' GCA TTT AAT GCA TTG ATG CC 3' The following primers are used: Nsi: 5 'GCA TTT AAT GCA TTG ATG CC 3'
Ch : 5' TTT CTC GAG TTA ACG GTA TTT GGA 3' Ch: 5 'TTT CTC GAG TTA ACG GTA TTT GGA 3'
D : 5' GTG CCT TTA GTG ATG CCG TGG CGC ACG TGG D: 5 'GTG CCT TTA GTG ATG CCG TGG CGC ACG TGG
ACG ACA TGC C 3' ACG ACA TGC C 3 '
D inv : 5' TCC ACG TGC GCC ACG GCA TCA CTA AAG GCA D inv: 5 'TCC ACG TGC GCC ACG GCA TCA CTA AAG GCA
C 3' C 3 '
Le fragment codant pour CIIFX NH-(1-73)bêta est amplifié à partir du vecteur pAT Pr bêta en utilisant les amorces Nsi et Dinv. Les conditions sont les suivantes : The fragment coding for CIIFX NH- (1-73) beta is amplified from the vector pAT Pr beta using the primers Nsi and Dinv. The conditions are as follows:
Tampon PCR 5 μl 5 μl PCR buffer
Mix 12 μl Mix 12 μl
Amorce Nsi 5 μi (à 10 μM) 5 μi Nsi primer (at 10 μM)
Amorce Dinv 5 μl (à 10 μM) Dinv primer 5 μl (at 10 μM)
Plasmide pAT Pr bêta 10 ng (1 μl) PAT Pr beta 10 ng (1 μl) plasmid
Taq polymérase (Cetus) 1 μl Taq polymerase (Cetus) 1 μl
H2O qsp 50 μl H 2 O qs 50 μl
On réalise 27 cycles : 27 cycles are performed:
97° C : 30 sec 97 ° C: 30 sec
58° C : 1 mn 58 ° C: 1 min
70° C : 2 mn = (2 sec/cycle) Le produit de PCR est précipité par Téthanol après extraction au phénol/chloroforme. Le fragment correspondant à la taille recherchée est recueilli par électroélution après électrophorèse sur gel d'agarose. 70 ° C: 2 min = (2 sec / cycle) The PCR product is precipitated with tethanol after extraction with phenol / chloroform. The fragment corresponding to the desired size is collected by electroelution after electrophoresis on agarose gel.
Le fragment codant pour alpha(69-141 )COOH de l'alpha globine est amplifié à partir du plasmide alphalpJW101 (Wilson et αl, 1978) en utilisant les amorces D et Ch. Les conditions sont les suivantes : Tampon PCR 5 μl The fragment coding for alpha (69-141) COOH of alpha globin is amplified from the plasmid alphalpJW101 (Wilson and αl, 1978) using primers D and Ch. The conditions are as follows: 5 μl PCR buffer
Mix 12 μl Mix 12 μl
Amorce D 5 μl (à 10 μM) Primer D 5 μl (at 10 μM)
Amorce Chιm6 5 μl (à 10 μM) Primer Chιm6 5 μl (at 10 μM)
Plasmide alphal 1 μl (= 10 ng) Alphal plasmid 1 μl (= 10 ng)
Taq polymérase (Cetus) 1 μl Taq polymerase (Cetus) 1 μl
H2O qsp 50 μl H 2 O qs 50 μl
On réalise 27 cycles de la même manière que pour la première amplification. 27 cycles are carried out in the same way as for the first amplification.
Il est purifié comme précédemment. It is purified as before.
Une troisième amplification est effectuée à partir des deux fragments purifiés en utilisant les amorces Nsi et Ch. Les conditions sont les suivantes : A third amplification is carried out from the two purified fragments using the primers Nsi and Ch. The conditions are as follows:
Tampon PCR 5 μl 5 μl PCR buffer
Mix 12 μl Mix 12 μl
Amorce Chim6 5 μl (à 10 μM) Chim6 primer 5 μl (at 10 μM)
Amorce Nsi 5 μl (à 10 μM) Nsi primer 5 μl (at 10 μM)
ADN obtenu par la DNA obtained by the
première PCR 1 μl (environ 50 ng) first PCR 1 μl (approximately 50 ng)
ADN obtenu par la DNA obtained by the
deuxième PCR 3 μl (environ 50 ng) second 3 μl PCR (approximately 50 ng)
Taq polymérase (Cetus) 1 μl Taq polymerase (Cetus) 1 μl
H2O qsp 50 μl H 2 O qs 50 μl
On effectue 35 cycles We perform 35 cycles
97° C : 30 sec 97 ° C: 30 sec
55° C : 1 mn 55 ° C: 1 min
70° C : 2 mn + (2 sec/cycle) Le produi t de PCR est précipité par Téthanol après extraction au phénol/chloroforme, puis purifié. Il est inséré dans le plasmide pAT Pr Tet aux sites Nsi l et Xho l . Ce vecteur permet d'exprimer une protéine de fusion comprenant : 70 ° C: 2 min + (2 sec / cycle) The PCR product is precipitated with tethanol after extraction with phenol / chloroform, then purified. It is inserted into the plasmid pAT Pr Tet at the Nsi 1 and Xho 1 sites. This vector makes it possible to express a fusion protein comprising:
- les 31 acides aminés N-terminaux de la protéine Cil du phage lambda,- the 31 N-terminal amino acids of the C11 protein of phage lambda,
- la séquence Ile-Glu-Gly-Arg reconnue par le facteur Xa, - the Ile-Glu-Gly-Arg sequence recognized by factor Xa,
- une sous-unité chimérique de 146 acides aminés, constituée par les résidus N terminaux 1 à 73 inclus de la chaîne bêta de globine et les résidus C terminaux 69 à 141 de la chaîne alpha (chimère NH 2(1 -73)bêta-alpha(69-141 )COOH). La figure 9 schématise les étapes de la construction. - a chimeric subunit of 146 amino acids, consisting of the N terminal residues 1 to 73 inclusive of the globin beta chain and the terminal C residues 69 to 141 of the alpha chain (NH 2 chimera (1-73) beta) alpha (69-141) COOH). Figure 9 shows schematically the stages of construction.
Ce plasmide est exprimé chez E. coli, souche CAG 1 1 39 (Grossman et αl, 1983, Cell, 32, 1 51 - 159). Cette souche de phénotype : F- thi leu thr LacY TonA SupE galK Lon 100 est transformée par la méthode conventionnelle de traitement au CaCl2. This plasmid is expressed in E. coli, strain CAG 1139 (Grossman et αl, 1983, Cell, 32, 1 51-159). This phenotype strain: F- thi leu thr LacY TonA SupE galK Lon 100 is transformed by the conventional method of treatment with CaCl 2 .
La culture est effectuée en milieu LB en présence de tetracycline 50 μg/ml à 30° C ; quand la DO atteint 0,8-1,2, l'expression de la protéine d'ADN est induite par passage à 42° C. Après 2 heures, il est possible de mettre en évidence la synthèse de la chaîne (bêta-alpha) sur gel de SDS-PAGE coloré au bleu de Coomassie par comparaison avec un échantillon de culture de CAG 1 139 non transformée. On estime que la quantité en chaîne bêta-alpha atteint 20 % des protéines totales. La construction a été séquencée pour vérifier qu'il n'y avait pas de mutation lors des PCR successifs. EXEMPLE 9: Caracterisation fonctionnelle des hémoglobines recombinantes The culture is carried out in LB medium in the presence of tetracycline 50 μg / ml at 30 ° C; when the OD reaches 0.8-1.2, the expression of the DNA protein is induced by passage at 42 ° C. After 2 hours, it is possible to demonstrate the synthesis of the chain (beta-alpha ) on SDS-PAGE gel stained with Coomassie blue by comparison with an untransformed CAG 1139 culture sample. It is estimated that the amount of beta-alpha chain reaches 20% of total proteins. The construction was sequenced to verify that there was no mutation during the successive PCRs. EXAMPLE 9 Functional Characterization of the Recombinant Hemoglobins
Mesures dans les conditions d'équilibre: Equilibrium measurements:
Après purification des solutions d'Hb, les spectres d'absorbance sont enregistrés et comparés au spectre de THb A native oxygénée (spectrophotomètre Cary 219, Var ian, Palo Alto, Ca, USA). Ces spectres permettent de tester la présence de méthémoglobine ou d'hémichrome (Szebeni et al,After purification of the Hb solutions, the absorbance spectra are recorded and compared to the spectrum of native oxygenated THb A (Cary 219 spectrophotometer, Varian, Palo Alto, Ca, USA). These spectra make it possible to test the presence of methemoglobin or of hemichrome (Szebeni et al,
1 984). Les mesures de l'affinité pour Toxygène à l'équilibre sont effectuées à l'aide d'un système automatique commercial (Hemox analyzer, TCS, Southampton PA, USA) (Asakura, 1979) associant la mesure simultanée de Tabsorbance à deux longueurs d'onde par spectrophotométπe et celle de la pression partielle de Toxygène par polarographie à l'aide d'une électrode à oxygène (YSI 5331 ). Dans les conditions habituelles les courbes de désoxygénation sont enregistrées dans des solutions d'Hb (60 à 100 μM en hème) dans un tampon 50 mM bisTris, 100 mM NaCl, pH 7,2 à 25°C, 50 μM d'EDTA et 20 μg/ml de cataiase sont ajoutés au tampon pour limiter l'oxydation de l'hémoglobine. La concentration de NaCl, le pH peuvent être modifiés pour tester les différentes propriétés de l'Hb recombinante (effet Bohr, effecteurs anioniques). D'autres effecteurs naturels (2,3 diphosphoglycérate) ou non naturels (IHP, dérivés du clofibrate) peuvent être ajoutés à la solution au début de l'enregistrement. Typiquement la solution est d'abord équilibrée sous un mélange gazeux dont la pression partielle en oxygène est d'au moins 350-450 mmHg pour assurer une saturation initiale quasi-complète de l'hémoglobine. La solution est alors désoxygénée lentement en admettant de l'azote pur dans la cuvette de mesure. La durée de l'enregistrement est de 45 à 60 min pour assurer les conditions d'équilibre. Ces conditions d'équilibre sont démontrées par la superposition des courbes de désoxygénation et de ré-oxygénation. La concentration d'Hb est mesurée après transformation en cyanmethémoglobine (epsilon540 = i l mM-1 cm-1 ). La quantité de methémoglobine (Hb oxydée) formée pendant l'enregistrement est mesurée par spectrophotométrie selon la méthode de Benèsh et ai (1973). 1,984). Affinity measurements for Toxygene at equilibrium are carried out using a commercial automatic system (Hemox analyzer, TCS, Southampton PA, USA) (Asakura, 1979) combining the simultaneous measurement of Tabsorbance at two lengths d wave by spectrophotometry and that of the partial pressure of Oxygen by polarography using an oxygen electrode (YSI 5331). Under the usual conditions, the deoxygenation curves are recorded in solutions of Hb (60 to 100 μM in heme) in a 50 mM bisTris buffer, 100 mM NaCl, pH 7.2 at 25 ° C., 50 μM of EDTA and 20 μg / ml of cataiase are added to the buffer to limit the oxidation of hemoglobin. The concentration of NaCl, the pH can be modified to test the different properties of recombinant Hb (Bohr effect, anionic effectors). Other natural (2,3 diphosphoglycerate) or non-natural (IHP, clofibrate derivatives) effectors can be added to the solution at the start of the recording. Typically the solution is first balanced under a gas mixture whose partial oxygen pressure is at least 350-450 mmHg to ensure an almost complete initial saturation of the hemoglobin. The solution is then slowly deoxygenated by admitting pure nitrogen into the measuring cuvette. The duration of the recording is 45 to 60 min to ensure the equilibrium conditions. These equilibrium conditions are demonstrated by the superposition of the deoxygenation and re-oxygenation curves. The Hb concentration is measured after transformation into cyanmethemoglobin (epsilon 540 = il mM -1 cm -1 ). The amount of methemoglobin (oxidized Hb) formed during the recording is measured by spectrophotometry according to the method of Benèsh et ai (1973).
200 à 300 couples de valeurs (Abs/PO2) sont en général enregistrés et stockés sur bande magnétique à l'aide d'un calculateur. Les valeurs de P50 (pO2 de demi-saturation et index de l'affinité pour Toxygène) et n 50 (index de la coopérativité) sont calculés à partir des mesures enregistrées entre 40 et 60 % de saturation par régression linéaire selon l'équation empirique de Hill ; l'ensemble des données des courbes d'équilibre sont ajustées à l'équation du modèle allosterique à deux états quaternaires (Monod et al, 1965) à l'aide de l'algorithme de régression itérative non-linéaire par moindres carrés (Bevington, 1969) comme décrit en détail dans Kister. et al, ( 1987). 200 to 300 pairs of values (Abs / PO2) are generally recorded and stored on magnetic tape using a computer. The values of P 50 (pO 2 half-saturation and the affinity index for Toxygene) and No. 50 (of the cooperativity index) are calculated from the measurements recorded between 40 and 60% saturation by linear regression according to the Hill's empirical equation; all the data of the equilibrium curves are fitted to the equation of the allosteric model with two quaternary states (Monod et al, 1965) using the regression algorithm nonlinear iterative by least squares (Bevington, 1969) as described in detail in Kister. et al, (1987).
La vitesse d'auto-oxydation de l'hémoglobine est mesurée par spectrophotométπe de la solution ( 1 00 μM hème) incubée dans un tampon 100 mM Phosphate à 37° C en fonction du temps (0—> 24 h). La stabilité thermique est mesurée par incubation des solutions (300 μM hème) à 65° C en fonction du temps (0—> 120 min). The auto-oxidation speed of hemoglobin is measured by spectrophotometry of the solution (1 00 μM heme) incubated in a 100 mM Phosphate buffer at 37 ° C. as a function of time (0-> 24 h). The thermal stability is measured by incubation of the solutions (300 μM heme) at 65 ° C as a function of time (0—> 120 min).
Mesures cinétiques Kinetic measurements
Les mesures cinétiques sont effectuées dans les solutions d'Hb recombinantes équilibrées sous 1 ou 0,1 atm de monoxyde de carbone (CO). Dans ces études on mesure spécifiquement la vitesse de recombinaison du CO à l'hémoglobine après sa photodisssociation par un éclair de lumière laser (Marden et αl, 1988). Kinetic measurements are carried out in recombinant Hb solutions balanced under 1 or 0.1 atm of carbon monoxide (CO). In these studies, the rate of CO recombination with hemoglobin is specifically measured after its photodisssociation by a flash of laser light (Marden and αl, 1988).
Les études de photodissociation sont pratiquées à l'aide d'un système laser 10-ns (Quaritel YG 585) délivrant 160 mJ à 532 nm. L'énergie du faisceau de photodissociation peut être modifiée pour étudier les conditions de photolyse partielle. Photodissociation studies are performed using a 10-ns laser system (Quaritel YG 585) delivering 160 mJ at 532 nm. The energy of the photodissociation beam can be modified to study the conditions of partial photolysis.
Les échantillons sont étudiés dans des cuvettes de 1 ou 2 mm de trajet optique. Le faisceau de détection à 436 nm est quasi cohnéaire avec le faisceau de photodissociation. L'intensité de lumière transmise est détectée par un photomultipiicateur (lP28 Hamamatsu, Japon) et enregistrée sur un oscilloscope digital (Lècroy 9400). Les données sont ensuite transférées dans un microcalculateur IBM pour les calculs des changements d'absorbance et l'analyse des données. Les simulations cinétiques sont basées sur le modèle allosterique à deux états. Typiquement la courbe enregistrée pour l'Hb A native montre l'existence de deux vitesses de recombinaison du CO à l'hémoglobine: Tune est rapide et correspond à la recombinaison à l'état R de forte affinité pour le ligand ; la deuxième est plus lente et correspond à la recombinaison à l'état T de faible affinité. Les expériences de cinétiques permettent de tester très rapidement la fonctionalité des Hb recombinantes avec le minimum d'échantillon. En présence d'hémine, les sous-unités chimériques obtenues selon les exemples s'assemblent en tétramère qui fixe l'oxygène de façon réversible pratiquement sans coopérativité mais avec une affinité légèrement inférieure à celle observée pour l'Hb A native et trente fois inférieure à celle du tétramère β 4. The samples are studied in cuvettes with 1 or 2 mm optical path. The detection beam at 436 nm is almost co-linear with the photodissociation beam. The intensity of transmitted light is detected by a photomultipiifier (lP28 Hamamatsu, Japan) and recorded on a digital oscilloscope (Lècroy 9400). The data is then transferred to an IBM microcomputer for calculations of absorbance changes and data analysis. Kinetic simulations are based on the two-state allosteric model. Typically the curve recorded for native Hb A shows the existence of two rates of recombination of CO to hemoglobin: Tune is rapid and corresponds to recombination in the R state of high affinity for the ligand; the second is slower and corresponds to recombination in the T state of low affinity. Kinetic experiments make it possible to very quickly test the functionality of recombinant Hb with the minimum of sample. In the presence of hemin, the chimeric subunits obtained according to the examples assemble in a tetramer which fixes the oxygen in a reversible manner practically without cooperativity but with an affinity slightly lower than that observed for native Hb A and thirty times lower to that of the β 4 tetramer.
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB8711614D0 (en) * | 1987-05-16 | 1987-06-24 | Medical Res Council | Proteins |
EP0402300B1 (en) * | 1989-05-10 | 1996-09-11 | Somatogen Inc. | Production of bacteria and yeast of hemoglobin and analogues thereof |
WO1991013158A1 (en) * | 1990-02-28 | 1991-09-05 | Delta Biotechnology Limited | Protein production in yeast |
US5239061A (en) * | 1990-06-20 | 1993-08-24 | Research Corporation Technologies, Inc. | Modified human hemoglobin, blood substitutes containing the same, and vectors for expressing the modified hemoglobin |
-
1992
- 1992-03-18 FR FR9203224A patent/FR2688784B1/en not_active Expired - Fee Related
-
1993
- 1993-03-18 CA CA002132345A patent/CA2132345A1/en not_active Abandoned
- 1993-03-18 AU AU48090/93A patent/AU4809093A/en not_active Abandoned
- 1993-03-18 EP EP93920553A patent/EP0633895A1/en not_active Withdrawn
- 1993-03-18 JP JP5516327A patent/JPH07507543A/en active Pending
- 1993-03-18 WO PCT/FR1993/000273 patent/WO1993019089A2/en not_active Application Discontinuation
Also Published As
Publication number | Publication date |
---|---|
FR2688784B1 (en) | 1995-06-30 |
CA2132345A1 (en) | 1993-09-30 |
JPH07507543A (en) | 1995-08-24 |
EP0633895A1 (en) | 1995-01-18 |
WO1993019089A3 (en) | 1993-10-28 |
AU4809093A (en) | 1993-10-21 |
FR2688784A1 (en) | 1993-09-24 |
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