WO1996002629B1 - Process for releasing heterolog proteins of saccharomyces cerevisiae strains - Google Patents
Process for releasing heterolog proteins of saccharomyces cerevisiae strainsInfo
- Publication number
- WO1996002629B1 WO1996002629B1 PCT/ES1995/000088 ES9500088W WO9602629B1 WO 1996002629 B1 WO1996002629 B1 WO 1996002629B1 ES 9500088 W ES9500088 W ES 9500088W WO 9602629 B1 WO9602629 B1 WO 9602629B1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- release
- strains
- heterologous proteins
- procedure
- proteins
- Prior art date
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract 26
- 102000004169 proteins and genes Human genes 0.000 title claims abstract 22
- 238000000034 method Methods 0.000 title claims abstract 17
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract 12
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 title claims 10
- 230000003834 intracellular effect Effects 0.000 claims abstract 9
- 230000003204 osmotic effect Effects 0.000 claims abstract 9
- 230000035772 mutation Effects 0.000 claims abstract 7
- 230000001413 cellular effect Effects 0.000 claims abstract 4
- 230000035945 sensitivity Effects 0.000 claims abstract 4
- 238000002360 preparation method Methods 0.000 claims abstract 3
- 230000035939 shock Effects 0.000 claims 5
- 239000002028 Biomass Substances 0.000 claims 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims 2
- 101150104869 SLT2 gene Proteins 0.000 claims 2
- 230000002358 autolytic effect Effects 0.000 claims 2
- 230000006037 cell lysis Effects 0.000 claims 2
- 230000002950 deficient Effects 0.000 claims 1
- 238000011534 incubation Methods 0.000 claims 1
- 238000002955 isolation Methods 0.000 claims 1
- 239000007788 liquid Substances 0.000 claims 1
- 239000000463 material Substances 0.000 claims 1
- 230000000050 nutritive effect Effects 0.000 claims 1
- 239000003381 stabilizer Substances 0.000 claims 1
- 238000005119 centrifugation Methods 0.000 abstract 1
- 230000009089 cytolysis Effects 0.000 abstract 1
- 239000012535 impurity Substances 0.000 abstract 1
- 238000000746 purification Methods 0.000 abstract 1
Abstract
The invention relates to a process for releasing intracellular heterolog proteins in the Saccharomyces Cerevisiae yeast based on the osmotic sensitivity which confers the mutation slt2 at the temperature of 37 °C. Said osmotic sensitivity may be used for releasing the intracellular content either by change of temperature of the culture from 24° to 37° C, or by transfer of grown cells in an osmotically stabilized medium at said second temperature to a non-stabilized medium. In both cases, the cells undergo a lysis and release their intracellular content and, after centrifugation to eliminate the cellular rests, a preparation of proteins is obtained which is appropriate to initiate a process of purification of the protein of interest. This process results in a cost reduction since the use of equipment is avoided and the preparation of proteins which is obtained contains less impurities than that which is obtained through other processes.
Claims
1.- Procedimiento de liberación, en porcentajes superiores al 50%, de proteínas heterólogas y otros materiales intracelulares (como productos particulados tipo VLP) producidos en Saccharomyces cerevisiae, basado en el empleo de estirpes mutadas en genes que afectan a la integridad celular, estables en condiciones de crecimiento normales de laboratorio (24- , medios nutritivos sencillos), y capaces de liberar el contenido intracelular por expresión de la correspondiente mutación en condiciones no permisivas.1.- Procedure for the release, in percentages higher than 50%, of heterologous proteins and other intracellular materials (such as VLP type particulate products) produced in Saccharomyces cerevisiae, based on the use of mutated strains in genes that affect cell integrity, stable under normal laboratory growth conditions (24-, simple nutritive media), and able to release the intracellular content by expression of the corresponding mutation under non-permissive conditions.
2.- Procedimiento de liberación de proteínas heterólogas producidas en cepas imitantes de Saccharomyces cerevisiae según la reivindicación número 1, estables en las condiciones de crecimiento adecuadas para la generación de una elevada biomasa celular.2. Process for the release of heterologous proteins produced in mutant strains of Saccharomyces cerevisiae according to claim 1, stable in the growth conditions suitable for the generation of a high cellular biomass.
3.- Procedimiento de liberación de proteínas heterólogas de cepas de3.- Procedure for the release of heterologous proteins from strains of
Saccharomyces cerevisiae según la reivindicación número 1 basado en la sensibilidad osmótica que confieren a las células mutaciones como las que afectan al gen SLT2 a temperaturas no permisivas como la de 37*C, y que determinan lisis celular de mas del 50% de las células en cultivo. .Saccharomyces cerevisiae according to claim 1 based on the osmotic sensitivity conferred on the cell mutations such as those affecting the SLT2 gene at non-permissive temperatures such as 37 * C, and which determine cell lysis of more than 50% of the cells in culture. .
4.- Procedimiento de liberación de proteínas heterólogas de cepas de Saccharomyces cerevisiae según las reivindicaciones números 1 y 3 caracterizado porque la obtención de preparaciones de proteínas heterólogas y productos particulados intracelulares se realiza por cualquier técnica que, produciendo la expresión de la mutación correspondiente, conduzca a la pérdida de integridad celular por la sensibilidad osmótica de las células.4. Procedure for the release of heterologous proteins from strains of Saccharomyces cerevisiae according to claims 1 and 3, characterized in that the preparation of heterologous proteins and intracellular particulate products is carried out by any technique which, producing the expression of the corresponding mutation, leads to to the loss of cellular integrity due to the osmotic sensitivity of the cells.
5.- Procedimiento de liberación de proteínas heterólogas de cepas de Saccharomyces cerevisiae según la reivindicación número 4 caracterizado porque la expresión de las mutaciones puede provocarse en las células capaces de expresar proteínas heterólogas o productos intracelulares particulados (es decir transformadas con el gen o genes correspondientes) mediante choque térmico de cultivos, es decir elevación de la temperatura hasta niveles no permisivos en medios no protegidos osmóticamente, o - β5. Procedure for the release of heterologous proteins of strains of Saccharomyces cerevisiae according to claim 4, characterized in that the expression of the mutations can be caused in cells capable of expressing heterologous proteins or intracellular particulate products (ie transformed with the corresponding gene or genes) ) by thermal shock of cultures, ie elevation of the temperature to non-permissive levels in osmotically unprotected media, or - β
por choque osmótico de células incubadas a temperatura no permisiva en medio osmóticamente protegido.by osmotic shock of cells incubated at non-permissive temperature in osmotically protected medium.
6.- Procedimiento para la obtención de proteínas heterólogas intracelulares y productos particulados caracterizado por el uso de otras especies de levadura diferentes de Saccharomyces cerevisiae en mutantes autolíticos, afectados en genes homólogos del gen SLT2 o en otros cuya mutación determine pérdida de la integridad celular, usando un choque osmótico o un choque térmico. 6.- Procedure for obtaining intracellular heterologous proteins and particulate products characterized by the use of other yeast species other than Saccharomyces cerevisiae in autolytic mutants, affected in homologous genes of the SLT2 gene or in others whose mutation determines loss of cellular integrity, using an osmotic shock or a thermal shock.
1 91 9
DECLARACIÓN SEGÚN EL ARTICULO 19DECLARATION ACCORDING TO ARTICLE 19
La originalidad de la invención creemos que está asegurada puesto que la publicación en que se describe su utilización para la obtención de proteínas heterólogas ( "A new systemfor the reléase of heterologous proteins from yeast based on mutant strains deficient in cell integrity". Alvarez et al. 1994. Journal of Biotechnology 5: 81-88) apareció con posterioridad suficiente a la solicituc" de patente nacional.The originality of the invention we believe is assured since the publication describes its use for obtaining heterologous proteins ("A new system for the release of heterologous proteins from yeast based on mutant strains deficient in cell integrity." Alvarez et al. 1994. Journal of Biotechnology 5: 81-88) appeared sufficiently after the national patent application.
En la publicación previa de nuestro grupo ("Expression of mutations andprotein rel ase by yeast conditional autolytic mutants in batch and continuous cultures", de la Fuente et al. 1993. Applied Microbiology and Biotechnology 38: 763-770), se describe la liberación de proteínas homologas como paite de un estudio fenotípico de las cepas mutantes, pero para nada se demuestra su aplicación a proteínas heterólogas, aunque se apunte en la discusión la posibilidad de abordar este último desarrollo.In the previous publication of our group ("Expression of mutations and protein safety by yeast conditional autolytic mutants in batch and continuous cultures", de la Fuente et al., 1993. Applied Microbiology and Biotechnology 38: 763-770), the release is described of homologous proteins as a part of a phenotypic study of the mutant strains, but its application to heterologous proteins is not proven at all, although the possibility of addressing this last development is noted in the discussion.
Por tanto, el descubrimiento del procedimiento útil para liberar proteínas heterólogas reivindicado, no se había publicado previamente. Además, al describir la citada liberación de proteínas homologas no se utilizó el choque osmótico como forma de desencadenar la lisis celular. A mayor abundamiento, hubieron de mejorarse las cepas a emplear para la liberación heteróloga para favorecer la estabilidad proteica con respecto a las descritas en la publicación en Applied Microbiology and Biotechnology.Therefore, the discovery of the procedure useful for releasing claimed heterologous proteins had not been previously published. In addition, when describing the aforementioned release of homologous proteins, osmotic shock was not used as a way to trigger cell lysis. Furthermore, the strains to be used for the heterologous release had to be improved to favor protein stability with respect to those described in the publication in Applied Microbiology and Biotechnology.
Por lo que respecta al procedimiento de liberación de proteínas descrito en la publicación "Isolation of recombinant proteins from Saccharomyces cerevisiae by use of osmotically fragüe mutant strains" (M. Broker. 1994. Biotechniques 16:604-610), se puede afirmar que difiere notablemente del reivindicado en nuestra solicitud de patente. Esta diferencia afecta en general a todas las características pero nos podemos fijar especialmente en dos. Por un lado estas últimas (las descritas en Biotechniques 16:604-610) son cepas poco estables cuyo crecimiento es difícil ya que sólo tiene lugar en presencia de un estabilizador osmótico sea cual sea la temperatura de incubación (son cepas condicionales osmóticas). Esto contrasta con nuestras cepas (que son termosensibles) que se mantienen perfectamente en cultivo a temperaturas inferiores a 28°C, óptimas para el crecimiento de S.cerevisiae, sin ninguna precaución especial y que pueden crecer fácilmente, por tanto, en medio líquido hasta elevadas densidades de biomasa. Por otro lado, dicha publicación (Biotechniques 16:604-610) asegura que con su procedimiento nunca se logra la liberación de mas del 20% de la proteína recombinante intracelular al no Usarse mas que ese porcentaje de células, sin que los autores 2 0As regards the protein release procedure described in the publication "Isolation of recombinant proteins from Saccharomyces cerevisiae by use of osmotically fragüe mutant strains" (M. Broker, 1994. Biotechniques 16: 604-610), it can be said that it differs remarkably from the one claimed in our patent application. This difference affects in general to all the characteristics but we can fix especially in two. On the one hand, the latter (those described in Biotechniques 16: 604-610) are unstable strains whose growth is difficult since it only takes place in the presence of an osmotic stabilizer whatever the temperature of incubation (they are osmotic conditional strains). This contrasts with our strains (which are thermosensitive) that are perfectly maintained in culture at temperatures below 28 ° C, optimal for the growth of S.cerevisiae, without any special precautions and that can grow easily, therefore, in liquid medium until high biomass densities. On the other hand, said publication (Biotechniques 16: 604-610) ensures that with its procedure the release of more than 20% of the intracellular recombinant protein is never achieved by not using more than that percentage of cells, without the authors twenty
encuentren una explicación para este fenómeno. Esto contrasta también con la intensa liberación que se logra con nuestro procedimiento.find an explanation for this phenomenon. This also contrasts with the intense release that is achieved with our procedure.
En -unción de lo anteriormente expuesto, hemos modificado ligeramente las reivindicaciones y hemos añadido una, para especificar mejor las características específicas, novedosas y diferenciales del procedimiento reivindicado. In view of the foregoing, we have modified the claims slightly and have added one, to better specify the specific, novel and differential characteristics of the claimed procedure.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU30787/95A AU3078795A (en) | 1994-07-14 | 1995-07-14 | Process for releasing heterolog proteins of saccharomyces cerevisiae strains |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ESP9401536 | 1994-07-14 | ||
ES9401536A ES2092439B1 (en) | 1994-07-14 | 1994-07-14 | RELEASE PROCEDURE OF HETEROLOGICAL PROTEINS FROM STRAINS OF SACCHAROMYCES CEREVISIAE. |
Publications (3)
Publication Number | Publication Date |
---|---|
WO1996002629A2 WO1996002629A2 (en) | 1996-02-01 |
WO1996002629A3 WO1996002629A3 (en) | 1996-04-04 |
WO1996002629B1 true WO1996002629B1 (en) | 1996-05-23 |
Family
ID=8286899
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/ES1995/000088 WO1996002629A2 (en) | 1994-07-14 | 1995-07-14 | Process for releasing heterolog proteins of saccharomyces cerevisiae strains |
Country Status (3)
Country | Link |
---|---|
AU (1) | AU3078795A (en) |
ES (1) | ES2092439B1 (en) |
WO (1) | WO1996002629A2 (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9810442D0 (en) * | 1998-05-16 | 1998-07-15 | Univ Manchester | Yeast |
ES2274664B1 (en) * | 2004-04-30 | 2008-04-01 | Consejo Sup. Investig. Cientificas | BGS4DELTA AUTOLITIC LEAVES, PROCEDURE OF OBTAINING AND ITS APPLICATIONS IN PROTEIN EXPRESSION PROCEDURES. |
-
1994
- 1994-07-14 ES ES9401536A patent/ES2092439B1/en not_active Expired - Fee Related
-
1995
- 1995-07-14 AU AU30787/95A patent/AU3078795A/en not_active Abandoned
- 1995-07-14 WO PCT/ES1995/000088 patent/WO1996002629A2/en active Application Filing
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