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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
• • C—CHEMISTRY; METALLURGY
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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

• Thyroid cancers are common endocrine malignancies.
• One function of the thyroid gland is to store thyroglobulin as a source of thyroid hormones, and patients with thyroid cancers routinely have increased circulating levels of thyroglobulin. Detecting increased levels of circulating thyroglobulin produced by these cancerous cells is, therefore, an extremely valuable tool for the physician in the diagnosis of thyroid cancers.
• Thyroglobulin levels in the sera of thyroid cancer patients are routinely quantified using various agency-approved (e.g. FDA) immunoassays. Physicians frequently order these diagnostic immunoassays to determine thyroglobulin levels in their patients. Therefore it is not surprising that such a commonly ordered diagnostic test has produced a variety of immunoassays developed by many different companies for use in the clinical laboratory.
• FDA agency-approved
• thyroglobulin for use as a standard in immunoassays comes from cadavers or surgically removed human tissue.
• the heterogeneity of human-derived thyroglobulin isolates is a limitation which has not been solved.
• anti-thyroglobulin autoantibodies present in some thyroid cancer patients' sera can interfere with immunoassays that attempt to detect and quantify thyroglobulin.
• Human thyreoglobulin is often iodinated with up to four iodine atoms (on various tyrosine residues). Defects in thyroglobulin are known to be the cause of congenital hypothyroidism due to dyshormonogenesis type 3.
• T3 serum free triiodothyronine
• T4 free tetraiodo thyronine
• the ability to synthesize homogenous thyroglobulin in an expression system, its use in assays, and the ability to chemically control the amount of iodination on the homogenous thyroglobulin molecules could provide means of accurately comparing the results of various assays, allow the removal of autoantibodies that bind thyroglobulin allowing for more accurate diagnoses of diseases, and also potentially lead to the treatment of patients that suffer from these thyroglobulin implicated diseases with synthesized thyroglobulin conjugates.
• the present invention includes novel soybean derived human thyroglobulin, methods of producing human thyroglobulin in plants such as soybean, and novel diagnostic applications for the detection and stratification of endocrine malignancies including thyroid cancer and thyroiditis.
• the invention also includes the use of soybean-derived human thyroglobulin in affinity matrices to remove autoreactive anti-thyroglobulin antibodies from patient's sera prior to analyses.
• Fig. 1 shows a plant transformation vector of the present invention.
• Fig. 2 illustrates PCR amplification of thyroglobulin DNA from other transgenic soybean seeds.
• Fig. 3. represents two confocal images; one a control and the other demonstrating hTG accumulation in transgenic Tl cotyledon tissue.
• Fig. 4. presents an ORGENTEC thyroglobulin ELISA of total soluble protein isolated from the indicated transgenic soybean lines. Each number represents an isolate from an individual soybean seed from that particular line. Results are presented as mean absorbance values. Wild type (WT) soluble soybean protein was used as a negative control (- ). The positive control (Human) represents human-purified thyroglobulin (Calbiochem) which was added to wild type soluble soybean protein.
• Fig. 5 shows KRONUS thyroglobulin ELISA of total soluble protein isolated from the indicated transgenic soybean lines. Each number represents an isolate from an individual soybean seed from that particular line. Results are presented as mean absorbance values showing two different dilutions of soluble protein (i.e., 1 : 100 and 1 : 1000) from each seed. Wild type (WT) soluble soybean protein was used as a negative control (-). The positive control (+) represents human-purified thyroglobulin (Human) which was added to wild type soluble soybean protein.
• Fig. 6A represents Sephacryl S-300 HR gel filtration
• chromatography of soybean-derived thyroglobulin (solid line) and thyroid-purified thyroglobulin (hatched line). Fractions were collected during each gel filtration, and subjected to ELISA to detect the presence of human thyroglobulin. Results are presented as percent relative ELISA absorbance (readings at 450 nm) for fractions representing the appropriate void volumes. The peak elution volumes of a set of molecular weight protein standards used for column calibration are indicated.
• Fig. 6B shows a western analysis of eluted fractions. Equal amounts of protein from the indicated fractions were separated in 5% native gels and subjected to western analysis. The location of the dimeric (D) form of hTG is indicated by the arrow.
• Fig. 7 shows a Western analysis of T2 seed protein.
• the crude seed extracts (5 ⁇ g) from 10 random T2 progeny were separated in 5% native polyacrylamide gels and screened by western blot analysis for the presence hTG.
• Fig. 8 represents western quantification of recombinant hTG using the gel on the left.
• the indicated amounts of commercially purified hTG protein and seed extract protein (line 77-5) were incubated in SDS-sample buffer (in the absence of ⁇ - mercaptoethanol and boiling) to relax dimeric protein and resulting in the predominantly monomeric protein for visualization and quantification following western analysis.
• a densitometry curve was generated by scanning the gel image and is shown on the right.
• Fig. 9 illustrates recognition of soybean-derived and thyroid-isolated thyroglobulin by autoantibodies in sera from patients' and controls using an ELISA. Plates were coated with equal amounts of soybean-derived or thyroid-isolated thyroglobulin. The ability of autoantibodies from patients' sera to recognize each protein was determined using an ELISA. Results are presented as means of triplicate determinations (+ SD).
• Fig. 10 shows a comparison of nucleotide sequences between the soy- optimized hTg sequence and the wildtype hTg sequence.
• the soy-optimized sequence is the top sequence and the wildtype hTg sequence is the bottom sequence.
• Fig. 11 shows a wildtype plant adjacent to the transformed plant containing the expressed thyroglobulin gene.
• the present invention solves two of the most significant problems plaguing FDA- approved thyroglobulin immunoassays by expressing human thyroglobulin in transgenic soybean seeds. It is useful to note the unique advantages of this platform technology for recombinant protein expression.
• thyroglobulin for use in immunodiagnostic assays is solely isolated from human thyroid tissue homogenates. Purification of this protein from homogenates is time consuming, and can lead to variability in composition and purity between lots [6]. The large number of contaminating thyroid proteins present in thyroid homogenates complicates the purification of thyroglobulin from this tissue.
• thyroglobulin cannot be utilized due to the large mass of thyroglobulin and requirement for proper post-translational modification and folding.
• bacterial expression systems also generally lack the ability to glycosylate proteins which would generally preclude the use of bacterial vectors as a means of making an adequate thyroglobulin.
• Yeast is also not likely to be a suitable vector for the expression of thyroglobulin. Although, yeast often post- translationally modifies proteins and has the ability to glycosylate proteins, it often suffers the drawback of uneven glycosylation and poor yield.
• the present invention does not suffer from these drawbacks.
• the number of proteins present in soybean seeds is quite limited [22]. Due to the physico- chemical characteristics of soybean proteins, purification schemes are more simple and straightforward than those currently used for human thyroid tissue. Furthermore, soybean- derived thyroglobulin was engineered to express a 6X-histidine tag (discussed in more detail below) so that affinity chromatography might be used in possible purification schemes.
• soybean-derived thyroglobulin will simplify its purification.
• the FDA requires each lot of an analyte isolated from human tissue to be screened for the absence of transmissible agents (e.g. HIV, Hepatitis B, etc.). Since thyroglobulin is isolated from human thyroid glands, each lot must be screened for such infectious agents. This adds to the cost of production, and increases possible product liability.
• transmissible agents e.g. HIV, Hepatitis B, etc.
• Soybean-derived thyroglobulin poses no risk of spreading human transmissible diseases.
• the FDA will not require screening for human infectious agents which would never be present. Ultimately, this will reduce the cost of production and limit product liability.
• thyroglobulin isolated from human thyroids is its heterogeneous nature. Since thyroid glands contain "immature” and “mature” forms of thyroglobulin that vary significantly in glycosylation and iodination, different lots of thyroglobulin can be quite dissimilar in purity and composition. This fact is borne out in some of our results where thyroglobulin isolated from human thyroid glands runs as a relatively broad peak on molecular sizing chromatography columns (see Fig. 6).
• Soybean-derived thyroglobulin of the present invention is much more homogenous than thyroid-isolated thyroglobulin for the following reasons.
• soybeans do not contain enzymes for iodination, therefore all soybean-derived thyroglobulin will have no iodine present. This represents a significant advantage since all thyroglobulin molecules will be homogenous relative to iodine content. This will allow non-iodinated lots of purified thyroglobulin to be produced.
• simple and straightforward iodination reactions e.g. Iodination Beads, Pierce Chem.
• Co. can be carried out on lots of thyroglobulin to produce uniformly iodinated protein of the different thyroglobulin molecules if needed.
• the treatment of non-iodinated thyroglobulin with the correct molar ratio of iodine and the correct oxidizing conditions will lead to a uniform amount of the T3 (or the Tl, T2, or T4) isomer. This flexibility will allow "custom" iodination of soybean-derived thyroglobulin as needed for various applications.
• proteins within soybean seeds are uniformly glycosylated. Since the soybean seed has a protein storage function, the proteins present in seeds are "mature" with uniform glycosylation [22]. This property will also contribute to the homogenous nature of soybean- derived thyroglobulin. While soy protein glycosylation will differ from human, the antibodies used in FDA approved ELISAs recognize the protein backbone, not carbohydrate residues. The uniform glycosylation of soybean-derived thyroglobulin will contribute to overall protein homogeneity, but not affect the ability of antibodies to bind this protein.
• Soybean-derived thyroglobulin will provide an improved, "reference" standard for quantification of this protein using FDA approved ELISAs when compared to thyroid- purified thyroglobulin.
• the transgenic soybean lines of the present invention express up to ⁇ 1% of their total soluble protein as thyroglobulin. With the screening of additional transgenic soybean lines, it may be possible to identify higher expressers. Regardless, a soybean seed weighs approximately 150 milligrams and contains approximately 40% protein (i.e. 60 milligrams of total protein per seed). At 1% expression, this represents 0.6 milligrams of thyroglobulin per seed.
• a single acre of soybeans could therefore produce approximately 10 kilograms of thyroglobulin at less than 0.01 cents per milligram using conventional methods. Even when considering growth in Biosafety Level 2 greenhouses, the cost increase for production will be approximately 20 fold (i.e. less than 2 cents per milligram) [23].
• Soybean seeds are comprised of -40% protein and therefore represent an ideal avenue for transgenic protein production. Moreover, at expression levels of -1%, it will be possible to contain the growth of transgenic soybeans in Biosafety Level 2 greenhouses without the need to produce in open fields [23]. At production levels of 10 kilograms per acre, and with the potential for 3 growing seasons per year, there would be no need for expansive open field plantings. Secure greenhouse growth would provide containment, and be consistent with good manufacturing practices for production of an FDA approved analyte.
• thyroglobulin isolated from human thyroid cadaver or surgical tissue and used in diagnostic assays comes from foreign suppliers (e.g. B.R.A.H.M.S., Berlin, Germany; Cis Bio International, Gif-sur-Yvette, France; Iason, Graz-Seierberg, Austria; Orgentec, Mainz, Germany). Therefore the ability to produce thyroglobulin in transgenic soybean seeds in the United States would reduce dependence on foreign suppliers.
• Human thyroglobulin is encoded by an 8.3 kb mRNA species encoding 2767 amino acids with a molecular weight of the mature monomer being over 300,000 daltons [4, 5].
• thyroglobulin is a very large protein which presents some significant challenges when trying to express this protein using traditional expression systems (e.g. E. coli) and it has been difficult (if not impossible) to accomplish.
• Yeast has also been used as a recombinant expression system.
• variations in glycosylation in yeast have been an obstacle that has often led to decreased yields and to the inventor's knowledge, yeast has not been capable of expressing thyroglobulin.
• thyroid gland One function of the thyroid gland is to store thyroglobulin [4, 5]. In this sense, the thyroid gland is a storage organ. Soybean seeds also function to store proteins needed for germination.
• a soybean compatible version of full-length human thyroglobulin was designed using criteria described in the art [24, 25].
• This "planticized" version of the human thyroglobulin gene was modified to optimize its expression in transgenic soybeans; however, it should be noted that the protein sequence encoded by this synthetic gene is identical to that of the human protein sequence. It was necessary to modify the nucleotide sequence, while keeping the encoded amino acids the same, to permit the soybean seeds to express optimal levels of this protein.
• hTg human Thyroglobulin
• ER endoplasmic reticulum
• KDEL lys-asp-glu-leu sequence
• the cloned synthetic gene could be placed downstream of the 7 S promoter and fused to a translational enhancer sequence (e.g. TEV, Tobacco Etch Virus).
• a translational enhancer sequence e.g. TEV, Tobacco Etch Virus
• His tag was added at the C-terminus.
• Other amino acid sequences to aid in purification were contemplated, such as GST tags, FLAG tags, HA tags, and MYC tags.
• biotin-strepavidin chemical tags can be used to aid in the purification process.
• the amino acid sequence of the expressed gene was cross checked against the updated sequence in the prior art [ 4].
• the second amino acid was chosen, except for 1819D and 251 IR.
• the inventors postulated and used 5' Ncol and 3' Xbal for cloning.
• the inventors did not use the TGA for the stop codon as the inventors knew that the overlapping methylation would prevent Xbal digestion.
• the wobble position of each codon was often changed to make the sequence more amenable to expression in soybean.
• the nucleotide sequence that is optimized for soy tends to contain a lower GC content than the corresponding wildtype human thyroglobulin.
• the nucleotide sequence was synthesized using standard nucleotide synthetic techniques by GeneArt (Burlingame, California) employing the strategy outlined above.
• a comparison between the open reading frame of wildtype thyroglobulin and the nucleotide sequence used for the soybean transformed thyroglobulin is shown in Fig. 10.
• the top sequence (query) represents the soy-optimized hTG and the wildtype human hTG is the bottom sequence (Sbjct).
• the top sequence representing the soy-optimized hTG is also SEQ ID NO: 1.
• the bottom sequence representing wildtype hTG is SEQ ID NO: 2.
• Wildtype human thyroglobulin has a plurality of exon regions (at least 48) that are post-translationally excised out.
• the soy-optimized SEQ ID NO: 1 was compared to the open reading frame of the wildtype thyroglobulin because it (i.e., SEQ ID NO: 2) (a) does not contain the exon sequences, (b) was recently updated to correct nucleotide errors and omissions, and (c) represented a consensus from a large number of individual sequences.
• the His tag (a plurality of CAT codons) at the 3' end of the nucleotide sequence in SEQ ID NO: 1 (i.e., the top sequence in Fig. 10).
• the His tag allows purification of the soy-optimized thyroglobulin by using a Ni + column or any other column that is able to preferentially bind a series of histidine residues.
• the synthetic hTG gene was designed and engineered as above to contain a native signal sequence, a GC content representative of plant systems, and codons that were optimized for expression in the Glycine max system.
• the synthetic hTG was subcloned downstream of the soybean ⁇ -conglycinin promoter resulting in the binary vector pPTN-hTG as shown in Fig. 1.
• the expression cassette was designed to contain P-7S (the soybean ⁇ -conglycinin promoter), TEV (tobacco etch virus translational enhancer element), and T-35S (cauliflower mosaic virus terminator element).
• the plant selection cassette contained P-nos (nopaline synthase promoter), Bar (phosphinothricin acetyltransferase gene for plant selection), and T-nos (nopaline synthase terminator element). Both cassettes were placed between the RB (right border sequence) and LB (left border sequence), in a binary vector that contained the aad A region (streptomycin resistance gene for bacterial selection).
• Soybean transformation using the Agrobacterium-mediated half seed method was performed as described in Paz et al (30). Briefly, half-seed explants (Glycine max) were dissected and inoculated with Agrobacterium suspension culture (strain EHAlOl carrying various binary vectors). The inoculated explants were placed adaxial side down on cocultivation medium at 24°C and under 18:6 photo period for 3-5 days. After cocultivation, explants were cultured for shoot induction and elongation under glufosinate selection (8 mg/L) for 8-12 weeks. Herbicide resistant shoots were harvested, elongated and rooted as described (30). Acclimated plantlets were transferred to soil and grown to maturity in the greenhouse. Transformation resulted in a total of five independent glufosinate-resistant events.
• Agrobacterium-mediated transformation resulted in five independent TO lines designated 77-3, 77-4, 77-5, 77-7 and 77-12. Phenotypically, TO parent plants as well as Tl and T2 progeny plants all appeared similar to wild type nontransgenic control plants with respect to leaf color, growth habit and relative seed yield (see Fig. 11). 60-day old transgenic (line 77-5) and WT (control) plants are shown in Fig. 11. To monitor for expression of the glufosinate herbicide selectable marker, Tl and T2 plants were sprayed with Ignite 280 SL herbicide (Bayer CropScience, RTP, NC) at a concentration of 80 mg/1 for a total of three times (days 1, 3, and 5). Plants with visible chlorosis similar to that observed in
• nontransgenic plants were scored as negative for resistance to the herbicide and discarded, while positive plants were taken to maturity. Plants known to be resistant to phosphinothricin were included as a control for spray concentration and application.
• genomic DNA was isolated from individual Tl seed shavings and from control seeds.
• genomic DNA was prepared from cotyledon tissue using the Maxwell 16 Instrument and Maxwell Tissue DNA Purification Kit (Promega, Madison, WI). Soybean genomic DNA (100 ng), TG primers (forward: 5'-GCTCAACCACTTAGACCATGCGA-S '; reverse: 5'- TCAGCGCAGTGGCAATATCCTG-3 '), vsp primers (forward: 5'-
• Soybean-derived thyroglobulin protein is recognized by commercially available ELISAs To begin to evaluate thyroglobulin protein expression by transgenic soybean seeds, two different commercially available ELISAs and one designed by the inventors were used. All of these ELISA use pairs of antibodies in a capture/detection format.
• the total soluble protein was isolated from 6 different individual Tl seed shavings from 5 different transgenic soybean lines.
• seed chips (10 mg of cotyledon tissue) were resuspended in 150 ⁇ l of phosphate buffered saline (PBS) and sonicated for 30 seconds using a Vibra-Cell ultrasonic processor (Newton, CT).
• PBS phosphate buffered saline
• Vibra-Cell ultrasonic processor Newton, CT.
• Samples were clarified from insoluble debris by centrifugation at 16.1 x 10 3 g at 4 0 C.
• Total soluble protein was quantified with the Bradford Reagent (Bio-Rad, Hercules, CA) using bovine serum albumin (BSA) as a standard.
• BSA bovine serum albumin
• Fig. 4 shows the results of one such assay from the Orgentec assay. It was clear from this ELISA that seeds from 4 of the 5 lines tested contained immunoreactive-thyroglobulin (line 77-4 did not contain the immunoreactive-thyroglobulin). The fact that this ELISA detected human thyroglobulin in sera strongly suggested that these particular transgenic soybean seeds expressed this protein.
• the commercially available ELISA from Orgentec uses polyclonal anti-human thyroglobulin antibodies to capture and detect human thyroglobulin. Such polyclonal antibodies likely bind both linear and conformational epitopes along the length of the thyroglobulin molecule.
• a more stringent test to evaluate the nature of soy-derived thyroglobulin would be the use of a second ELISA procedure which utilizes monoclonal antibodies for capture and detection, respectively.
• the commercially available ELISA produced by Kronus, Inc. (Boise, ID) is such an assay, and employs monoclonal antibodies which can simultaneously recognize two different conformational determinants on human thyroglobulin. This assay was used to detect the presence of thyroglobulin in selected soy protein samples that were identified as expressing this protein in Fig. 4 above.
• the Organtec ELISA uses polyclonal antibodies for detection of thyroglobulin.
• a second commercially-available kit for detecting thyroglobulin is the Kronus ELISA. While the Orgentec kit uses two polyclonal antibodies for detection, the Kronus kit utilizes separate monoclonal antibodies for detection.
• Fig. 5 shows the results of one such assay. It was clear from this ELISA that the seeds tested contained immunoreactive-thyroglobulin. The fact that this ELISA uses two monoclonal antibodies to capture and detect thyroglobulin provides further support for the authenticity of soybean-derived thyroglobulin.
• Fig. 5 shows the Kronus ELISA used for detection of hTG in select Tl seed extracts. Crude seed extract from one representative Tl progeny (indicated by an asterisk in Fig. 4) was examined, along with soluble protein from a nontransgenic seed (WT) and commercially-purified hTG. Five different dilutions of each selected sample were tested in the ELISA and absorbance values for two of these dilutions (1 : 100 and 1 : 1000) are shown along with controls for comparison.
• a third sandwich-based ELISA was developed and this ELISA utilized a monoclonal antibody for capture and a polyclonal antibody for detection. Briefly, 500 ng of capture antibody (GTX21984, GeneTex, Irvine, CA) was coated onto ELISA plates by incubation at 4 0 C for 16 hours. Unbound antibody was washed with PBS and nonspecific binding sites were blocked by incubation with 1% BSA in PBS for 1 hour at 23 0 C. Soy protein samples and the hTG standard were then loaded onto plates and allowed to complex with the bound antibody for 2 hours at 23oC.
• capture antibody GTX21984, GeneTex, Irvine, CA
• human thyroid-purified thyroglobulin (Calbiochem, Inc.) was diluted in 0.5 ml of wild type soy protein, and applied to the same column. Eluted fractions were also collected.
• Thyroglobulin is approximately 330 kDa as a monomer, but exists in solutions as a 660 kDa dimer. Therefore it was of interest to determine whether soybean-derived thyroglobulin could also form dimers. Both thyroglobulin protein preparations had a peak elution volume similar to that observed for bovine thyroglobulin (at 669 kDa). In fact, it appears that soybean-derived thyroglobulin was somewhat more homogenous in its elution profile than that observed for human thyroid-purified thyroglobulin (as the peak is sharper - see Fig. 6A).
• soybean-derived thyroglobulin could form ⁇ 660 kDa dimers, strongly suggesting that this protein folds in a manner similar to thyroid-isolated human thyroglobulin, allowing dimer formation.
• a sephacryl S-300 HR gel filtration column (bed height 72 cm) was calibrated by determining the peak elution volumes (absorbance at 254 nm,
• BioLogic LP BIO-RAD, Inc.
• molecular weight protein standards Sigma, Inc.
• Crude, total soluble protein was then isolated from hTG-positive seeds, and applied to a gel filtration column, and eluted fractions were collected.
• human thyroid-purified thyroglobulin was applied to the same column, and eluted fractions were also collected.
• Eluted fractions were then subjected to ELISA (Orgentec) to detect the presence of immunoreactive thyroglobulin in each fraction.
• thyroglobulin could form 660 kDa dimers (see Fig. 6B). This result suggested that monomers would have a size of approximately 330 kDa.
• protein extracts from transgenic and wild type seeds were run in 5% native poly aery lamide gels for approximately 2 hours at 1 10V. Unless noted, neither the gel, sample buffer nor running buffer contained ⁇ - mercaptoethanol or SDS, and samples were not boiled prior to loading onto the gel. Purified hTG (EMD Chemicals, Gibbstown, NJ) was included as a standard. Following
• Membranes were then incubated with goat anti-rabbit HRP(horse radish peroxidase)-conjugated IgG (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 minutes at 23 0 C and washed. Detection was carried out using the SuperSignal West Pico substrate (Thermo Scientific, Rockford, IL).
• gel filtration chromatography was used as shown in Fig. 6A to partially purify proteins from crude soluble seed extracts.
• a Sephacryl S-300 HR gel filtration column was calibrated by determining the peak elution volumes of a commercial set of molecular mass standards ranging in size from 669 kDa to 29 kDa. The largest of these molecular mass standards was bovine thyroglobulin (MW -669 kDa) and eluted in fraction 20.
• ⁇ -amylase is the standard at 443 kDa and alcohol dehydrogenase is the standard at 200 kDa.
• transgenic seed extract from line 77-5 was applied to the Sephacryl column, and the eluted protein in each fraction was subjected to an ELISA for detection of hTG.
• the immunoreactive profile for soy-derived hTG is shown as a solid line in Fig. 6A. Although fractions 17-23 contained detectable levels of hTG, the peak immunoreactivity was localized to fractions 20 and 21. Fractions 1-11 and 28-36 showed minimal absorbance and therefore are not included on the plot in Figure 6A.
• Fig. 3 shows the results of confocal microscopy which was performed as follows. Whole seed tissue was imbibed for 16 hours in IX PBS and the seed coat was removed. Tissue was fixed as described previously by our laboratory (25, 31). Briefly, sections were permeabilized with IX PBS containing 0.2% Tween for 10 minutes, and nonspecific binding was blocked by incubation with IX PBS supplemented with 3% BSA for 4 hours at 23 0 C. Tissue was incubated with rabbit anti-hTG serum (1:20 dilution) for 16 hours at 4 0 C, followed by incubation with an AlexaFluor 594 goat anti-rabbit IgG-HRP conjugated secondary antibody (1:200 dilution) for 1 hour at 23 0 C.
• DAPI 6-diamidino-2-phenylindole
• Cover slips were added to the sections using Gel/Mount aqueous mounting media. Images were collected with a LSM 710 Spectral Confocor 3 Confocal Microscope (Carl Zeiss, Inc.) using a 40X objective and a 405 nm laser to visualize DAPI stained nuclei, along with a 561 nm laser to collect emitted fluorescence from the Alexafluor 594 antibody. Stacks of images (30 optical sections, 17 nm apart) were collected in the Z plane of the specimens and projected to form a single image. To improve clarity and reproduction quality, image colors were proportionally enhanced using the ZEN 2009 Light Edition software.
• the lighter color (shows as blue if image is in color) at the nuclei of the cells represents DAPI staining while the lighter color (shows as red if image is in color) on the outer surface of the cells represents fluorescence from the AlexaFluor antibody recognizing transgenic hTG.
• Figs 7 and 8 For western visualization and quantification, as shown in Figs 7 and 8, known amounts of commercially-purified hTG protein and crude seed-extracted protein (line 77-5) were incubated with SDS-sample buffer lacking ⁇ -mercaptoethanol, and electrophoresed in 5% native polyacrylamide gels. Western blots were performed and X-ray films of the resulting blots were scanned for densitometric analysis. Integrated density was measured using ImageJ software (Rasband, 1997-2005). The image was inverted and background pixel values were subtracted. A standard curve was plotted using these integrated density values and the known amounts of purified hTG protein, from which an absolute value of hTG in the seed sample was determined.
• hTG O.Olng- 10ng
• crude seed extracted protein 10-fold dilutions over four orders of magnitude
• Absorbance values from the known concentrations of hTG were used to generate a curve, and the concentrations of hTG in seed extracts was determined by extrapolation of hTG concentration for those samples with absorbance values falling within the linear range of the curve. Absolute values were converted to a percentage of total protein.
• a western analysis of T2 seed protein is shown.
• the crude seed extracts (5 ⁇ g) from 10 random T2 progeny were separated in 5% native polyacrylamide gels and screened by western blot analysis for the presence hTG.
• Thyroid-purified hTG (75 ng) served as a standard to visualize monomeric and dimeric forms of the protein.
• Nontransgenic seed protein (5 ⁇ g) served as a negative control.
• Fig. 8 the western quantification of recombinant hTG is shown.
• the indicated amounts of commercially purified hTG protein and seed extract protein (line 77-5) were incubated in SDS-sample buffer (in the absence of ⁇ -mercaptoethanol and boiling) to relax dimeric protein and result in predominantly monomeric protein for visualization and quantification following western analysis.
• a densitometry curve was generated by scanning the gel image and plotting integrated density of each known standard using ImageJ software. Extrapolation from this curve revealed 27.97 ng of hTG protein in 5 ug of seed extract, representing 0.6% of total soluble seed protein for ths particular sample.
• thyroid-isolated thyroglobulin (Calbiochem, Inc.) or soybean- derived thyroglobulin were separately fractionated on a Sephacryl S-300 HR gel filtration column in a manner similar to that shown in Fig. 6A.
• fractions representing 59 to 60 milliliters of column void volume for thyroid-isolated and soybean- derived thyroglobulin were concentrated (using a Centricon-100). Quantification of the concentrated protein was accomplished using Bradford assays. Equivalent amounts (100 ng/well) of each thyroglobulin preparation were coated onto ELISA microtiter plates (Nunc high-binding) overnight as is routine in our laboratory.
• Fig. 9 shows that regardless of the source of thyroglobulin used to coat plates, there was no significant difference in the ability of autoantibodies in patients' sera to recognize soybean-derived (solid bars) or thyroid-isolated (open bars) thyroglobulin. These results further demonstrate the antigenic identity of these two thyroglobulin isolates and suggest that the soybean derived thyroglobulin is similar to if not identical to at least one conformer of human wild type thyroglobulin.
• the present invention relates to novel soy-optimized thyroglobulin sequences, transformed plants, methods of making and using the Soy-optimized thyroglobulin sequences such as assays using the Soy-optimized thyroglobulin sequence(s).
• SEQ ID NO: 3 is nucleotides 1-279 of SEQ ID NO: 1
• SEQ ID NO: 4 is nucleotides 280-558 of SEQ ID NO: 1, etc.
• the present invention relates to a transgenic plant transformed with an exogenous nucleotide sequence that expresses a protein or a domain thereof wherein said protein or domain thereof is thyroglobulin or a domain thereof.
• the exogenous nucleotide sequence(s) comprises one or more sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:
• the transgenic plant is a soybean plant.
• SEQ ID NO: 1 represents the full length nucleotide sequence that expresses full length soy optimized thyroglobulin.
• the other SEQ ID numbers are different regions of the full length nucleotide sequence that expresses full length soy optimized thyroglobulin (i.e., SEQ ID NO: 1).
• SEQ ID NO: 1 represents the full length nucleotide sequence that expresses full length soy optimized thyroglobulin.
• SEQ ID NO: 2 is different regions of the full length nucleotide sequence that expresses full length soy optimized thyroglobulin (i.e., SEQ ID NO: 1).
• the present invention relates to a transgenic plant transformed with an exogenous nucleotide sequence that expresses a protein where the protein is thyroglobulin and the exogenous nucleotide sequence is SEQ ID NO: 1.
• the present invention relates to a transgenic soybean wherein the exogenous nucleotide sequence comprises one or more sequences selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16.
• the exogenous nucleotide sequence comprises one or more sequences selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.
• the transgenic soybean further comprises a nucleotide sequence that codes for a sequence that allows protein purification and/or a nucleotide sequence that confers selection (such as antibiotic resistance and/or herbicidic resistance and/or provides some distinguishing characteristic to the soybean plant that allows transgenic soybeans to be differentiated from wild type soybean plants) to the transgenic soybean.
• the selection gene can be either operably linked to the gene or it can be introduced in an "unlinked” fashion. This means that the transgenic soybean can be segregated away to leave only the gene of interest (e.g. hTG) in the transgenic plant.
• Possible protein purification sequences include one or more of a His tagged linker, GST tags, FLAG tags, HA tags, and MYC tags. It is also contemplated that chemical binding reagents can be used in purification procedures. For example, biotin may be attached to the thyroglobulin protein or domain thereof allowing purification using strepavidin.
• the transgenic soybean may possess a plant selectable marker such as a sequence that confers antibiotic and/or herbicidic resistance.
• this resistance may be glufosinate resistance.
• other sequences that confer antibiotic and/or herbicidic resistance may be used.
• the transgenic plant (soybean) of the present invention may have a gene itself, or there may be a gene in a bacterial vector that is used in the plant that provides a selectable marker such as conferring resistance to antibiotics and/or herbicides.
• Non-limiting examples include a gene that confers ampicillin resistance, cloaxicillin resistance, kanamycin resistance, or bialophos resistance.
• a screenable marker may be used instead of, or in conjunction with a selectable marker.
• the transgenic soybean will overexpress the soy optimized thyroglobulin such that the thyroglobulin or domain thereof is expressed in an amount that is 0.3% or greater of total protein concentration.
• the level of protein may be 0.5% or greater of total protein concentration.
• the level of protein may be 1% or greater of total protein concentration.
• the level of protein may be 2% or greater of total protein concentration.
• the level of protein may be 3% or greater of total protein concentration.
• Soybean can be readily transformed by an array of different transformation methods which have been developed and optimized over the past decade in various laboratories. In addition to the above described transformations, other transformations known in the art may be used.
• the present invention might use transformation techniques that are the cotyledonary node transformation using the bacteria Agrobacterium tumefaciens or the particle bombardment of somatic embryogenic cultures. Regeneration using somatic embryogenesis is also within the scope of the present invention using a variety of explant tissue including embryonic axes, intact zygotic embryos, and excised cotyledons.
• soybean transformation Other methods have also been developed to transform soybean and are contemplated and therefore within the scope of the present invention.
• One example is the introduction of exogenous DNA into a plant embryo through the pollen tube pathway after pollination.
• Another method is the use of Agrobacterium rhizogenes, which causes hairy root disease and is used in a manner similar to A. tumefaciens to infect wound sites on roots and transfer T- DNA from the bacterial cell to the plant cell.
• Other methods of soybean transformation include electroportation, microinjection, silicon carbide fibers, liposome-mediated transformation and in planta Agrobacterium-mediated transformation using vacuum infiltration of whole plants.
• the present invention relates to an isolated nucleic acid comprising SEQ ID NO: 1 and variants thereof that are at least 80% identical to SEQ ID NO: 1.
• the nucleic acid of SEQ ID NO: 1 and variants thereof may be purified.
• the isolated nucleic acid may comprise SEQ ID NO: 1 and variants thereof that are at least 90% identical to SEQ ID NO: 1.
• the isolated nucleic acid may comprise SEQ ID NO: 1 and variants thereof that are at least 92% identical to SEQ ID NO: 1.
• the isolated nucleic acid may comprise SEQ ID NO: 1 and variants thereof that are at least 95% identical to SEQ ID NO: 1.
• the isolated nucleic acid may comprise SEQ ID NO: 1 and variants thereof that are at least 99% identical to SEQ ID NO: 1.
• the present invention relates to an isolated nucleic acid having a nucleotide sequence selected from the group consisting of a) the nucleotide sequence set forth in SEQ ID NO: 1 and b) a fragment of the nucleotide sequence of SEQ ID NO: 1 that is at least 100 nucleotides in length.
• the fragment of the nucleotide sequence of SEQ ID NO: 1 may be at least 50 nucleotides in length.
• the fragment of the nucleotide sequence of SEQ ID NO: 1 may be at least 250 nucleotides in length.
• the fragment of the nucleotide sequence of SEQ ID NO: 1 may be at least 500 nucleotides in length.
• the present invention relates to a nucleic acid comprising one or more sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.
• the present invention relates to a method of producing and isolating human thyroglobulin or a domain thereof comprising: a) synthesizing an exogenous nucleotide sequence comprising one or more sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32;
• SEQ ID NO: 24 SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32; c) growing the soybean in a medium wherein human thyroglobulin or a domain thereof is expressed from one or more of said sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27,
• the transformed soybean further comprises one or more of a seed-specific promoter(s), a nucleotide sequence expressing a transcriptional or translational enhancer (e.g. tobacco etch virus), a polyadenylation signal, a His tag nucleotide sequence, a nucleotide sequence that confers antibiotic resistance, and/or a leader sequence (or endogenous leader or any alternative leader sequence that targets expression to the secretory pathway (for example in the ER).
• a seed-specific promoter(s) e.g. tobacco etch virus
• a nucleotide sequence expressing a transcriptional or translational enhancer e.g. tobacco etch virus
• a polyadenylation signal e.g. tobacco etch virus
• His tag nucleotide sequence e.g. a polyadenylation signal
• a leader sequence or endogenous leader or any alternative leader sequence that targets expression to the secretory pathway (for example in the ER).
• the transformed soybean comprises a leader sequence and this transformed soybean may be used in the above method(s).
• an expression cassette is used and is cloned into a binary vector.
• the expression cassette cloned into the binary vector may be used in the transgenic soybean or in any method using and/or making the transgenic soybean.
• IVD In Vitro Diagnostic
• Soy-Tg such as an Analyte-Specific Reagent (ASR).
• ASR Analyte-Specific Reagent
• IPD in vitro diagnostic
• thyroiditis patients can be screened for the presence of serum autoantibodies against thyroglobulin (TG). The presence of such autoantibodies confirms disease, suggests treatment regimens, and aids in monitoring development of disease.
• TG thyroglobulin
• thyroid cancer patients who have had organ ablation, are routinely monitored for the presence of serum TG to assess whether malignant thyroid tissue still remains.
• these various IVD immunoassays are the gold standards for diagnosis and monitoring.
• Soy-Tg can be used in IVD assays that 1) detect antibodies against
• Human-Tg and 2) require a reference standard for quantification of Human Tg levels in fluids (for example, including but not limited to its use in sera, saliva, urine, blood, etc.).
• Soy-Tg can be used as a reference standard for the industry.
• the lack of a universally consistent Human-Tg standard contributes significantly to variability between FDA-approved assays made by different suppliers (see 14, 15, 16, 17 and 18). Differences in the quantification of thyroglobulin between assays are so great that the results from one assay cannot be extrapolated to results obtained using other assays.
• Soy-Tg Soy-Tg or domains thereof can be used as the reference standard for the industry to normalize all assays using hTg.
• Soy-Tg can be used in connection with a medical device to remove anti-thyroglobulin (TG) antibodies from biological samples (e.g. patients' sera) to eliminate interference in hTg immunoassays.
• TG anti-thyroglobulin
• hTg immunoassays The most significant limitation with present day hTg immunoassays is the quantification of thyroglobulin levels in patients' sera that contain autoantibodies against thyroglobulin (see 16 and 18). Detection of thyroglobulin in cancer patients' sera that have had thyroid ablation is the gold standard for detecting metastasized tumor tissue.
• the present invention relates to a method of accurately detecting thyroglobulin concentration or a concentration of a domain of thyroglobulin in an individual comprising: a) obtaining sera from the individual; b) contacting the sera with an immunoaffinity disc that comprises thyroglobulin or a domain thereof isolated from a transformed soybean; c) allowing the immunoaffinity disc that comprises thyroglobulin or the domain thereof to bind antibodies in the sera; d) separating the sera from the immunoaffinity disc; and e) detecting the amount of thyroglobulin in the sera.
• the method can be used to detect cancer, wherein an elevated thyroglobulin concentration indicates the presence of cancer.
• the method uses thyroglobulin protein or a domain thereof isolated from a transformed soybean, which is encoded for by an exogenous nucleotide sequence and said exogenous nucleotide sequence comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO:
• the immunoaff ⁇ nity disc that comprises
• thyroglobulin or a domain thereof is attached to the immunoaff ⁇ nity disc by a covalent bond.
• the bond may be ionic, or there may be other forces that keep the thyroglobulin or domain thereof attached to the disc, such as van der Waals forces and/or hydrophobic interactions.
• the soy optimized thyroglobulin is expressed free of iodine.
• iodine can be chemically added so that the amount of iodination is carefully controlled.
• the present invention relates to thyroglobulin expressed by the nucleotide sequences of the present invention with differing amounts of iodine present therein (e.g., TO, Tl, T2, T3, T4, etc.).
• the non-iodinated or iodinated thyroglobulin can be administered to an individual to treat the individual for one of the thyroglobulin implicated diseases.
• methods using non-iodinated and iodinated thyroglobulin for this treatment are contemplated.
• Clark PM Laboratory services for thyroglobulin and implications for monitoring of differentiated thyroid cancer. Journal of clinical pathology. 2009 May;62(5):402-6.
• Gagnon P The emerging generation of chromatography tools for virus purification.

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