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| Efficient Expression and Secretion of a Long-acting Form of Human Growth Hormone in Pichia pastoris |
| QI Nan1, ZHANG Yan-hong2, WU Jun2, MA Qing-jun2 |
1. Department of Biochemistry and Molecular Biology, Anhui Medical University, Hefei 230022, China);
2. Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100850, China |
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Abstract Objective: To prolong the half-life of human growth hormone (HGH) in the blood plasma, the engineering Pichia pastoris yeast strain of efficient expression and secretion of human serum albumin (HSA)-HGH fusion protein was constructed.Method: The genes of HSA and HGH were amplified respectively from human fetal liver cDNA library and HGH engineering strain vector. After cloned into the expression vector pHIL-D2, the recombinant plasmid was transformed into Pichia pastoris GS115 strain by electroporation. The efficient expression and secreting strain was screened out by in situ double filter screening method, and then confirmed respectively by SDS-PAGE and Western blotting. Its culture condition was researched. The fusion protein was purified from the culture of the yeast by ion exchange, affinity and gel filtration chromatography, and then confirmed by N terminal sequence test, molecular weight test and isoelectric focus electrophoresis. Finally, the pharmacokinetics and pharamacodynamics test of frozen dry protein powder were carried out in cynomolgus monkeys. Results: The best culture condition of engineering yeast was established, with the highest yield of 100 mg/L. The purity was over 95 percent, and the productivity was over 42 percent. The protein demonstrated good bioactivity by the cynomolgus mokeys test, and has a 6.8-fold longer half-life and a 44-fold slower clearance than equimolar doses of HGH. Conclusion: The fusion protein demonstrates a long acting characteristic, which suggests its promising application in clinical medicine.
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Received: 21 June 2012
Published: 25 December 2012
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[1] Kostyo J L, Cameron C M, Olson K C, et al. Biosynthetic 20-kilodalton methionyl-human growth hormone has diabetogenic and insulin-like activities. Proc Natl Acad Sci U S A, 1985, 82 (12): 4250-4253.
[2] Fares F, Guy R, Bar-Ilan A, et al. Designing a long-acting human growth hormone (hGH) by fusing the carboxyl-terminal peptide of human chorionic gonadotropin-subunit to the coding sequence of hGH. Endocrinology, 2010, 151 (9): 4410-4417.
[3] Grigorian A L, Bustamante J J, Hernandez P, et al. Extraordinarily stable disulfide-linked homodimer of human growth hormone. Protein Sci, 2005, 14 (4): 902-913.
[4] Linglart A, Cabrol S, Berlier P, et al. Growth hormone treatment before the age of 4 years prevents short stature in young girls with Turner syndrome. European Journal of Endocrinology, 2011, 164 (6): 891-897.
[5] Osborn B L, Sekut L, Corcoran M, et al. Albutropin: a growth hormone-albumin fusion with improved pharmacokinetics and pharmacodynamics in rats and monkeys. Eur J Pharmacol, 2002, 456 (1-3): 149-158.
[6] Masumoto N, Tateno C, Tachibana A, et al. GH enhances proliferation of human hepatocytes grafted into immunodeficient mice with damaged liver. Journal of Endocrinology, 2007, 194 (3): 529-537.
[7] Kenth G, Mergelas J A, Goodyer C G. Developmental changes in the human GH receptor and its signal transduction pathways. Journal of Endocrinology, 2008, 198 (1): 71-82.
[8] Dumas H, Panayiotopoulos P, Parker D, et al. Understanding and meeting the needs of those using growth hormone injection devices. BMC Endocr Disord, 2006, 6: 5.
[9] Brearley C, Priestley A, Leighton-Scott J, et al. Pharmacokinetics of recombinant human growth hormone administered by cool.clickTM 2, a new needle-free device, compared with subcutaneous administration using a conventional syringe and needle. BMC Clinical Pharmacology, 2007, 7 (1): 10.
[10] Chou D K, Krishnamurthy R, Randolph T W, et al. Effects of Tween 20 and Tween 80 on the stability of Albutropin during agitation. J Pharm Sci, 2005, 94 (6): 1368-1381.
[11] 王楠, 赵洪亮, 刘志敏, 等. 生长激素释放激素和人血清白蛋白融合蛋白的克隆表达. 生物技术通讯, 2007, 18 (4): 574-577. Wang N, Zhao H L, Liu Z M, et al. Cloning and expressing of HSA-GHRH fusion protein. Letters in Biotechnology, 2007, 18(4): 574-577.
[12] 唱韶红, 巩新, 杨志愉,等. 人血清白蛋白和人干扰素α2b的融合蛋白在毕赤酵母中的表达. 生物工程学报, 2006, 22(2): 173-179. Chang S H, Gong X, Yang Z Y, et al. Expression in Pichia pastoris and properties of human serum albumin-interferon alpha2b chimera. Chinese Biotechnology, 2006, 22 (2): 173-179.
[13] Bo H, Minjian L, Guoqiang H, et al. Expression of hepatitis B virus S gene in Pichia pastoris and application of the product for detection of anti-HBs antibody. J Biochem Mol Biol, 2005, 38 (6): 683-689.
[14] McGrew J T, Leiske D, Dell B, et al. Expression of trimeric CD40 ligand in Pichia pastoris: use of a rapid method to detect high-level expressing transformants. Gene, 1997, 187 (2): 193-200.
[15] Cos O, Ramon R, Montesinos J L, et al. Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: a review. Microb Cell Fact, 2006, 5: 17.
[16] Reiter E O, Attie K M, Moshang T, et al. A multicenter study of the efficacy and safety of sustained release GH in the treatment of naive pediatric patients with GH deficiency. J Clin Endocrinol Metab, 2001, 86 (10): 4700-4706.
[17] Melder R J, Osborn B L, Riccobene T, et al. Pharmacokinetics and in vitro and in vivo anti-tumor response of an interleukin-2-human serum albumin fusion protein in mice. Cancer Immunol Immunother, 2005, 54 (6): 535-547.
[18] Zhao T, Cheng Y N, Tan H N, et al. Site-specific chemical modification of human serum albumin with polyethylene glycol prolongs half-life and improves intravascular retention in mice. Biol Pharm Bull, 2012, 35 (3): 280-288.
[19] Marques J A, George J K, Smith I J, et al. A barbourin-albumin fusion protein that is slowly cleared in vivo retains the ability to inhibit platelet aggregation in vitro. Thromb Haemost, 2001, 86 (3): 902-908.
[20] Cereghino J L, Cregg J M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev, 2000, 24 (1): 45-66.
[21] Weidner M, Taupp M, Hallam S J. Expression of recombinant proteins in the methylotrophic yeast Pichia pastoris. Journal of Visualized Experiments, 2010, 36:1862.
[22] Weber H, Barth G. Nonconventional yeasts: their genetics and biotechnological applications. Crit Rev Biotechnol, 1988, 7 (4): 281-337.
[23] Sinha J, Plantz B A, Inan M, et al. Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovine interferon-tau. Biotechnol Bioeng, 2005, 89 (1): 102-112.
[24] Wang Z, Wang Y, Zhang D, et al. Enhancement of cell viability and alkaline polygalacturonate lyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation. Bioresour Technol, 2010, 101 (4): 1318-1323.
[25] Murphy K P, Gagne P, Pazmany C, et al. Expression of human interleukin-17 in Pichia pastoris: purification and characterization. Protein Expr Purif, 1998, 12 (2): 208-214. |
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