单链抗体的原核可溶性表达及临床应用_West China Medical Journal

Authors：

周勇军 ¹ , 姚于勤 ¹ ,  杨金亮 ¹ , 王明蓉 ²

Corresponding author：

杨金亮, Email: bioyjzhou@gmail.com

Keywords：

单链抗体; 可溶性表达; 大肠杆菌; 临床应用

DOI：

CNKI: 51-1356/R.20120115.1555.038

Video：

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利用原核表达系统表达外源基因来获取大量有活性的蛋白质是目前最常用且经济的方法。由于原核表达系统的自身特点，单链抗体的表达往往以无活性的包涵体存在，研究者为解决此问题探索了许多方法。现就在大肠杆菌中表达可溶单链抗体的策略以及临床应用研究作一综述。

Citation： 周勇军,姚于勤,杨金亮,王明蓉. 单链抗体的原核可溶性表达及临床应用. West China Medical Journal, 2012, 27(1): 136-139. doi: CNKI: 51-1356/R.20120115.1555.038 Copy

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, 王小宁. HBscFv-IFNγ在毕赤酵母X33中的表达、纯化及鉴定[J]. 生物工程学报, 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, 王小宁. HBscFv-IFNγ在毕赤酵母X33中的表达、纯化及鉴定[J]. 生物工程学报, 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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