單鏈抗體的原核可溶性表達及臨床應用_《華西醫學》

作者：

周勇軍 ¹ , 姚于勤 ¹ ,  楊金亮 ¹ , 王明蓉 ²

1四川大學生物治療國家重點實驗室（成都，610041）;2中國醫藥集團總公司四川抗菌素工業研究所;

關鍵詞：

單鏈抗體可溶性表達大腸桿菌臨床應用

DOI：

CNKI: 51-1356/R.20120115.1555.038

視頻：

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利用原核表達系統表達外源基因來獲取大量有活性的蛋白質是目前最常用且經濟的方法。由于原核表達系統的自身特點，單鏈抗體的表達往往以無活性的包涵體存在，研究者為解決此問題探索了許多方法。現就在大腸桿菌中表達可溶單鏈抗體的策略以及臨床應用研究作一綜述。

引用本文： 周勇軍,姚于勤,楊金亮,王明蓉. 單鏈抗體的原核可溶性表達及臨床應用. 華西醫學, 2012, 27(1): 136-139. doi: CNKI: 51-1356/R.20120115.1555.038 復制

1.	Schirrmann T, Al-Halabi L, Dübel S, et al. Production systems for recombinant antibodies[J]. Fronti Biosci, 2008, 13: 4576-4594.
2.	Jordan E, Hust, Roth A, et al. Production of recombinant antibody fragments in bacillus megaterium[J]. Microb Cell Fact, 2007, 6: 2.
3.	Austin C. Novel approach to obtain biologically active recombinant heterodimeric proteins in E. coli[J]. J Chromatogr B Ana-lyt Technol Biomed Life Sci, 2003, 786(1-2): 93-107.
4.	Kolaj O, Spada S, Robin S, et al. Use of folding modulators to improve heterologous protein production in Escherichia coli[J]. Microb Cell Fact, 2009, 8: 9.
5.	Makrides SC. Strategies for achieving high-level expression of genes in Escherichia coli[J]. Microbiol Rev, 1996, 60(3): 512-538.
6.	Schein CH, Boix E, Haugg M, et al. Secretion of mammalian ribonucleases from Escherichia-coli using the signal sequence of murine spleen ibonuclease[J]. Biochem J, 1992, 283(1): 137-141.
7.	Derman AI, Prinz WA, Belin D, et al. Mutations that allow disulfide bond formation in the cytoplasm of Escherichia-coli[J]. Science, 1993, 262(5140): 1744-1747.
8.	Jurado J, de Lorenzo V, Luis A, et al. Thioredoxin fusions Increase folding of single chain Fv antibodies in the cytoplasm of Escherichiacoli: evidence that chaperone activity is the prime effect of thioredoxin[J]. J Mol Biol, 2006, 357(1): 49-61.
9.	Dammeyer T, Steinwand M, Krüger SC, et al. Efficient production of soluble recombinant single chain Fv fragments by a Pseudomonas putida strain KT2440 cell factory[J]. Microb Cell Fact, 2011, 10: 11.
10.	Jurado P, Ritz D, Beckwith J, et al. Production of functional single-chain Fv antibodies in the cytoplasm of Escherichia coli[J]. J Mol Biol, 2002, 320(1): 1-10.
11.	Bessette PH, Aslund F, Beckwith J, et al. Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm[J]. Proc Natl Acad Sci USA, 1999, 96(24): 13703-13708.
12.	Venturi M, Seifert C, Hunte C. High level production of functional antibody Fab fragments in an oxidizing bacterial cytoplasm[J]. J Mol Biol, 2002, 315(1): 1-8.
13.	Bach H, Mazor Y, Shaky S, et al. Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies[J]. J Mol Biol, 2001, 12(1): 79-93.
14.	Zheng L, Baumann U, Reymond JL. Production of a functional catalyticantibody ScFv-NusA fusion protein in bacterial cytoplasm[J]. JBiochem, 2003, 133(5): 577-581.
15.	Sonoda H, Kumada Y, Katsuda T, et al. Functional expression of single-chain Fv antibody in the cytoplasm of Escherichia coli by thioredoxin fusion and co-expression of molecular chaperones[J]. Protein Expr Purif, 2010, 70(2): 248-253.
16.	Wang C, Castro AF, Wilkes DM, et al. Expression and purifcation of the first nucleotide-binding domain and linker region of human multidrug resistance gene product: comparison of fusions to glutathione S-transferase, thioredoxin and maltose-binding protein[J]. Biochem J, 1999, 338(1): 77-81.
17.	Bach H, Mazor Y, Shaky S, et al. Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies[J]. J Mol Biol, 2001, 312(1): 79-93.
18.	Hu X, O’Hara L, White S, et al. Optimisation of production of a domoic acid-binding scFv antibody fragment in Escherichia coli using molecular chaperones and functional immobilisation on a mesoporous silicate support[J]. Protein Expr Purif, 2007, 52(1): 194-201.
19.	Ow DS, Lim DY, Nissom PM, et al. Co-expression of Skp and FkpA chaperones improves cell viability and alters the global expression of stress response genes during scFvD1. 3 roduction[J]. Microb Cell Fact, 2010, 9: 22.
20.	Sonoda H, Kumada Y, Katsuda T, et al. Effects of cytoplasmic and periplasmic chaperones on secretory production of single-chain Fv antibody in Escherichia coli[J]. J Biosci Bioeng, 2011, 111(4): 465-470.
21.	Zhang Z, Li ZH, Wang F, et al. Overexpression of DsbC and DsbG markedly improves soluble and functional expression of single-chain Fv antibodies in Escherichia coli[J]. Protein Expr Purif, 2002, 26(2): 218-228.
22.	Mergulhão FJ, Monteiro GA. Periplasmic targeting of recombinant proteins in Escherichia coli[J]. Methods Mol Biol, 2007, 390: 47-61.
23.	Wang L, Miller A, Kendall DA. Signal peptide determinants of SecA binding and stimulation of ATPase activity[J]. J Biol Chem, 2000, 275(14): 10154-10159.
24.	Ostermeier M, De Sutter K, Georgiou G. Eukaryotic protein disul?de isomerase complements Escherichia coli dsbA mutants and increases the yield of a heterologous secreted protein with disulfide bonds[J]. J Biol Chem, 1996, 271(18): 10616-10622.
25.	Clark KR, Walsh ST. Crystal structure of a 3B3 variant-a broadly neutralizing HIV-1 scFv antibody[J]. Protein Sci, 2009, 8(12): 2429-2441.
26.	Zhang ZX, Lazdina U, Chen M, et al. Characterization of a monoclonal antibody and its single-chain antibody fragment recognizing the nucleoside Triphosphatase/Helicase domain of the hepatitis C virus nonstructural 3 protein[J]. Clin Diagn Lab Immunol, 2000, 7(1): 58-63.
27.	周世水, 王小寧. HBscFv-IFNγ在畢赤酵母X33中的表達、純化及鑒定[J]. 生物工程學報, 2008, 24(3): 423-429.
28.	Begent RH, Verhaar MJ, Chester KA, et al. Clinical evidence of efficient tumor targeting based on single-chain Fv antibody selected from a combinatorial library[J]. Nat Med, 1996, 2(9): 979-984.
29.	Benhar I, Reiter Y, Pai LH, et al. Administration of disulfide-stabilized Fv-immunoloxins Bl（dsFv）-PE38 by continuous infusion increases their efficacy in curing large tumor xenografts in nude mice[J]. Int J Cancer, 1995, 62(3): 351-355.
30.	George AJ, Jamar F, Tai MS, et al. Radiometal labeling of recombinant proteins by a genetically engineered minimal chelation site: technetium-99m coordination by single-chain Fv antibody fusion proteins through a C-terminal cysteinyl peptide[J]. Proc Natl Acad Sci USA, 1995, 92(18): 8358-8362.

1. 　Schirrmann T, Al-Halabi L, Dübel S, et al. Production systems for recombinant antibodies[J]. Fronti Biosci, 2008, 13: 4576-4594.
2. 　Jordan E, Hust, Roth A, et al. Production of recombinant antibody fragments in bacillus megaterium[J]. Microb Cell Fact, 2007, 6: 2.
3. 　Austin C. Novel approach to obtain biologically active recombinant heterodimeric proteins in E. coli[J]. J Chromatogr B Ana-lyt Technol Biomed Life Sci, 2003, 786(1-2): 93-107.
4. 　Kolaj O, Spada S, Robin S, et al. Use of folding modulators to improve heterologous protein production in Escherichia coli[J]. Microb Cell Fact, 2009, 8: 9.
5. 　Makrides SC. Strategies for achieving high-level expression of genes in Escherichia coli[J]. Microbiol Rev, 1996, 60(3): 512-538.
6. 　Schein CH, Boix E, Haugg M, et al. Secretion of mammalian ribonucleases from Escherichia-coli using the signal sequence of murine spleen ibonuclease[J]. Biochem J, 1992, 283(1): 137-141.
7. 　Derman AI, Prinz WA, Belin D, et al. Mutations that allow disulfide bond formation in the cytoplasm of Escherichia-coli[J]. Science, 1993, 262(5140): 1744-1747.
8. 　Jurado J, de Lorenzo V, Luis A, et al. Thioredoxin fusions Increase folding of single chain Fv antibodies in the cytoplasm of Escherichiacoli: evidence that chaperone activity is the prime effect of thioredoxin[J]. J Mol Biol, 2006, 357(1): 49-61.
9. 　Dammeyer T, Steinwand M, Krüger SC, et al. Efficient production of soluble recombinant single chain Fv fragments by a Pseudomonas putida strain KT2440 cell factory[J]. Microb Cell Fact, 2011, 10: 11.
10. 　Jurado P, Ritz D, Beckwith J, et al. Production of functional single-chain Fv antibodies in the cytoplasm of Escherichia coli[J]. J Mol Biol, 2002, 320(1): 1-10.
11. 　Bessette PH, Aslund F, Beckwith J, et al. Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm[J]. Proc Natl Acad Sci USA, 1999, 96(24): 13703-13708.
12. 　Venturi M, Seifert C, Hunte C. High level production of functional antibody Fab fragments in an oxidizing bacterial cytoplasm[J]. J Mol Biol, 2002, 315(1): 1-8.
13. 　Bach H, Mazor Y, Shaky S, et al. Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies[J]. J Mol Biol, 2001, 12(1): 79-93.
14. 　Zheng L, Baumann U, Reymond JL. Production of a functional catalyticantibody ScFv-NusA fusion protein in bacterial cytoplasm[J]. JBiochem, 2003, 133(5): 577-581.
15. 　Sonoda H, Kumada Y, Katsuda T, et al. Functional expression of single-chain Fv antibody in the cytoplasm of Escherichia coli by thioredoxin fusion and co-expression of molecular chaperones[J]. Protein Expr Purif, 2010, 70(2): 248-253.
16. 　Wang C, Castro AF, Wilkes DM, et al. Expression and purifcation of the first nucleotide-binding domain and linker region of human multidrug resistance gene product: comparison of fusions to glutathione S-transferase, thioredoxin and maltose-binding protein[J]. Biochem J, 1999, 338(1): 77-81.
17. 　Bach H, Mazor Y, Shaky S, et al. Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies[J]. J Mol Biol, 2001, 312(1): 79-93.
18. 　Hu X, O’Hara L, White S, et al. Optimisation of production of a domoic acid-binding scFv antibody fragment in Escherichia coli using molecular chaperones and functional immobilisation on a mesoporous silicate support[J]. Protein Expr Purif, 2007, 52(1): 194-201.
19. 　Ow DS, Lim DY, Nissom PM, et al. Co-expression of Skp and FkpA chaperones improves cell viability and alters the global expression of stress response genes during scFvD1. 3 roduction[J]. Microb Cell Fact, 2010, 9: 22.
20. 　Sonoda H, Kumada Y, Katsuda T, et al. Effects of cytoplasmic and periplasmic chaperones on secretory production of single-chain Fv antibody in Escherichia coli[J]. J Biosci Bioeng, 2011, 111(4): 465-470.
21. 　Zhang Z, Li ZH, Wang F, et al. Overexpression of DsbC and DsbG markedly improves soluble and functional expression of single-chain Fv antibodies in Escherichia coli[J]. Protein Expr Purif, 2002, 26(2): 218-228.
22. 　Mergulhão FJ, Monteiro GA. Periplasmic targeting of recombinant proteins in Escherichia coli[J]. Methods Mol Biol, 2007, 390: 47-61.
23. 　Wang L, Miller A, Kendall DA. Signal peptide determinants of SecA binding and stimulation of ATPase activity[J]. J Biol Chem, 2000, 275(14): 10154-10159.
24. 　Ostermeier M, De Sutter K, Georgiou G. Eukaryotic protein disul?de isomerase complements Escherichia coli dsbA mutants and increases the yield of a heterologous secreted protein with disulfide bonds[J]. J Biol Chem, 1996, 271(18): 10616-10622.
25. 　Clark KR, Walsh ST. Crystal structure of a 3B3 variant-a broadly neutralizing HIV-1 scFv antibody[J]. Protein Sci, 2009, 8(12): 2429-2441.
26. 　Zhang ZX, Lazdina U, Chen M, et al. Characterization of a monoclonal antibody and its single-chain antibody fragment recognizing the nucleoside Triphosphatase/Helicase domain of the hepatitis C virus nonstructural 3 protein[J]. Clin Diagn Lab Immunol, 2000, 7(1): 58-63.
27. 　周世水, 王小寧. HBscFv-IFNγ在畢赤酵母X33中的表達、純化及鑒定[J]. 生物工程學報, 2008, 24(3): 423-429.
28. 　Begent RH, Verhaar MJ, Chester KA, et al. Clinical evidence of efficient tumor targeting based on single-chain Fv antibody selected from a combinatorial library[J]. Nat Med, 1996, 2(9): 979-984.
29. 　Benhar I, Reiter Y, Pai LH, et al. Administration of disulfide-stabilized Fv-immunoloxins Bl（dsFv）-PE38 by continuous infusion increases their efficacy in curing large tumor xenografts in nude mice[J]. Int J Cancer, 1995, 62(3): 351-355.
30. 　George AJ, Jamar F, Tai MS, et al. Radiometal labeling of recombinant proteins by a genetically engineered minimal chelation site: technetium-99m coordination by single-chain Fv antibody fusion proteins through a C-terminal cysteinyl peptide[J]. Proc Natl Acad Sci USA, 1995, 92(18): 8358-8362.

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單鏈抗體的原核可溶性表達及臨床應用

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單鏈抗體的原核可溶性表達及臨床應用

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