Stability improvement of human collagen α1(I) chain using insulin as a fusion partner

doi:10.1016/j.cjche.2018.04.008

Chinese Journal of Chemical Engineering ›› 2018, Vol. 26 ›› Issue (12): 2607-2614.DOI: 10.1016/j.cjche.2018.04.008

• Biotechnology and Bioengineering • 上一篇下一篇

Stability improvement of human collagen α1(I) chain using insulin as a fusion partner

Yu Mi¹, Yuan Gao¹, Daidi Fan¹, Zhiguang Duan¹, Rongzhan Fu¹, Lihua Liang¹, Wenjiao Xue², Shanshan Wang³

1 Shaanxi R & D Center of Biomaterials and Fermentation Engineering, School of Chemical Engineering, Northwest University, Xi'an 710069, China;
2 Shaanxi Institute of Microbiology, Xi'an 710043, China;
3 School of Biological Science and Engineering, Shaanxi University of Technology, Hanzhong 723001, China

收稿日期:2017-12-20 修回日期:2018-03-24 出版日期:2018-12-28 发布日期:2019-01-09
通讯作者: Daidi Fan
基金资助:
Supported by the National Natural Science Foundation of China (21676214, 21576160, 21506171), Shaanxi Key Laboratory of Degradable Biomedical Materials Program (2014SZS07-K04, 2014SZS07-P05, 15JS105, 15JS106, 2014SZS07-Z01, 2014SZS07-K03), Shaanxi R&D Center of Biomaterials and Fermentation Engineering Program (2015HBGC-04).

Stability improvement of human collagen α1(I) chain using insulin as a fusion partner

Yu Mi¹, Yuan Gao¹, Daidi Fan¹, Zhiguang Duan¹, Rongzhan Fu¹, Lihua Liang¹, Wenjiao Xue², Shanshan Wang³

1 Shaanxi R & D Center of Biomaterials and Fermentation Engineering, School of Chemical Engineering, Northwest University, Xi'an 710069, China;
2 Shaanxi Institute of Microbiology, Xi'an 710043, China;
3 School of Biological Science and Engineering, Shaanxi University of Technology, Hanzhong 723001, China

Received:2017-12-20 Revised:2018-03-24 Online:2018-12-28 Published:2019-01-09
Contact: Daidi Fan
Supported by:
Supported by the National Natural Science Foundation of China (21676214, 21576160, 21506171), Shaanxi Key Laboratory of Degradable Biomedical Materials Program (2014SZS07-K04, 2014SZS07-P05, 15JS105, 15JS106, 2014SZS07-Z01, 2014SZS07-K03), Shaanxi R&D Center of Biomaterials and Fermentation Engineering Program (2015HBGC-04).

摘要/Abstract

摘要： To enhance the stability of recombinant human collagen α1(I) chains (rhCOL1A1) in production and purification stages, a gene fragment fusing COL1A1 and insulin protein coding domains was synthesized and inserted into the pPIC9K expression vector. The fusion peptide-expressing Pichia pastoris strain was created by transformation. After optimization of shake flask cultures, the ultimate intracellular expression level of the insulin-collagen α1 (I) chain fusion protein (INS-COL1A1) reached about 300 mg·L^-1, and no obvious protein degradation was found in the fermentation and purification processes. The His-tagged recombinant fusion protein was detected by western blotting and was effectively purified using Ni²⁺-chelating chromatography. A prominent improvement in the stability of INS-COL1A1 was observed compared to rhCOL1A1 in vitro, and the rhCOL1A1 released from the fusion protein was studied by LC-MS/MS and in bioassays. The results showed that the purified rhCOL1A1 was consistent with the native protein in amino acid composition and had a similar biological compatibility. To our knowledge, this is the first study to demonstrate the use of insulin as a fusion protein to improve the stability of easily degradable proteins.

关键词: Fusion expression, Human collagen &alpha, 1(I) chain, Insulin, Protein stability, Pichia pastoris

Abstract: To enhance the stability of recombinant human collagen α1(I) chains (rhCOL1A1) in production and purification stages, a gene fragment fusing COL1A1 and insulin protein coding domains was synthesized and inserted into the pPIC9K expression vector. The fusion peptide-expressing Pichia pastoris strain was created by transformation. After optimization of shake flask cultures, the ultimate intracellular expression level of the insulin-collagen α1 (I) chain fusion protein (INS-COL1A1) reached about 300 mg·L^-1, and no obvious protein degradation was found in the fermentation and purification processes. The His-tagged recombinant fusion protein was detected by western blotting and was effectively purified using Ni²⁺-chelating chromatography. A prominent improvement in the stability of INS-COL1A1 was observed compared to rhCOL1A1 in vitro, and the rhCOL1A1 released from the fusion protein was studied by LC-MS/MS and in bioassays. The results showed that the purified rhCOL1A1 was consistent with the native protein in amino acid composition and had a similar biological compatibility. To our knowledge, this is the first study to demonstrate the use of insulin as a fusion protein to improve the stability of easily degradable proteins.

Key words: Fusion expression, Human collagen &alpha, 1(I) chain, Insulin, Protein stability, Pichia pastoris

Yu Mi, Yuan Gao, Daidi Fan, Zhiguang Duan, Rongzhan Fu, Lihua Liang, Wenjiao Xue, Shanshan Wang. Stability improvement of human collagen α1(I) chain using insulin as a fusion partner[J]. Chinese Journal of Chemical Engineering, 2018, 26(12): 2607-2614.

参考文献

[1] K. Gelse, E. Poschl, T. Aigner, Collagens-Structure, function, and biosynthesis, Adv. Drug Deliv. Rev. 55(12) (2003) 1531-1546.

[2] S. Gomes, I.B. Leonor, J.F. Mano, L.R. Rui, D.L. Kaplan, Natural and genetically engineered proteins for tissue engineering, Prog. Polym. Sci. 37(1) (2012) 1-17.

[3] M.W. Werten, T.J. van den Bosch, R.D. Wind, H. Mooibroek, F.A. de Wolf, High-yield secretion of recombinant gelatins by Pichia pastoris, Yeast 15(11) (1999) 1087-1096.

[4] C. Rutschmann, S. Baumann, J. Cabalzar, K.B. Luther, T. Hennet, Recombinant expression of hydroxylated human collagen in Escherichia coli, Appl. Microbiol. Biotechnol. 98(10) (2014) 4445-4455.

[5] C. Zhang, J. Baez, K.M. Pappu, C.E. Glatz, Purification and characterization of a transgenic corn grain-derived recombinant collagen type I alpha 1, Biotechnol. Prog. 25(6) (2009) 1660-1668.

[6] M. Pizarro-Guajardo, V. Olguin-Araneda, J. Barra-Carrasco, C. Brito-Silva, M.R. Sarker, D. Paredes-Sabja, Characterization of the collagen-like exosporium protein, BclA1, of Clostridium difficile, spores, Anaerobe 25(1) (2014) 18-30.

[7] L.B. Li, D.D. Fan, X.X. Ma, J.J. Deng, J. He, High-level secretory expression and purification of unhydroxylated human collagen α1(Ⅲ) chain in Pichia pastoris GS115, Biotechnol. Appl. Biochem. 62(4) (2014) 467-475.

[8] L.N. Wang, D.D. Fan, J. He, Z.C. Lv, C.H. Zhu, A new strategy for secretory expression and mixed fermentation of recombinant human collagen α1(Ⅲ) chain in Pichia pastoris, Biotechnol. Bioprocess Eng. 19(5) (2014) 916-924.

[9] X. Xing, Q. Gan, R.C. Clough, K.M. Pappu, J.A. Howard, J.A. Baez, K. Wang, Hydroxylation of recombinant human collagen type I alpha 1 in transgenic maize coexpressed with a recombinant human prolyl 4-hydroxylase, BMC Biotechnol. 11(1) (2011) 69-80.

[10] D.M. Correia, J. Padrao, L.R. Rodrigues, F. Dourado, S. Lanceros-Mendez, V. Sencadas, Thermal and hydrolytic degradation of electrospun fish gelatin membranes, Polym. Test. 32(5) (2013) 995-1000.

[11] T.J. Magliery, L. Regan, Combinatorial approaches to protein stability and structure, Eur. J. Biochem. 271(9) (2004) 1595-1608.

[12] S. Adinolfi, M. Nair, A. Politou, E. Bayer, S. Martin, P. Temussi, A. Pastore, The factors governing the thermal stability of frataxin orthologues:how to increase a protein's stability, Biochemistry 43(21) (2004) 6511-6518.

[13] D.F. Steiner, S.Y. Park, J. Stoy, L.H. Philipson, G.I. Bell, A brief perspective on insulin production, Diabetes Obes. Metab. 11 Suppl. 4(s4) (2009) 189-196.

[14] D. Boyhan, H. Daniell, Low-cost production of proinsulin in tobacco and lettuce chloroplasts for injectable or oral delivery of functional insulin and C-peptide, Plant Biotechnol. J. 9(5) (2011) 585.

[15] A. Trabucchi, L.L. Guerra, N.I. Faccinetti, R.F. Iacono, E. Poskus, S.N. Valdez, Expression and characterization of human proinsulin fused to thioredoxin in Escherichia coli, Appl. Microbiol. Biotechnol. 94(6) (2012) 1565-1576.

[16] L.M. Damasceno, C.J. Huang, C.A. Batt, Protein secretion in Pichia pastoris, and advances in protein production, Appl. Microbiol. Biotechnol. 93(1) (2012) 31-39.

[17] C. Gurramkonda, S. Polez, N. Skoko, A. Adnan, T. Gabel, D. Chugh, S. Swaminathan, N. Khanna, S. Tisminetzky, U. Rinas, Application of simple fed-batch technique to highlevel secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin, Microb. Cell Factories 9(1) (2010) 1-11.

[18] Y. Wang, Z.H. Liang, Y.S. Zhang, S.Y. Yao, Y.G. Xu, Y.H. Tang, S.Q. Zhu, D.F. Cui, Y.M. Feng, Human insulin from a precursor overexpressed in the methylotrophic yeast Pichia pastoris and a simple procedure for purifying the expression product, Biotechnol. Bioeng. 73(1) (2001) 74-79.

[19] N.A. Baeshen, M.N. Baeshen, A. Sheikh, R.S. Bora, M.M.M. Ahmed, H.A.I. Ramadan, Cell factories for insulin production, Microb. Cell Factories 13(1) (2014) 141.

[20] T. Jiang, C. Kang, X.W. Yu, Y. Xu, High-level expression of prolyl endopeptidase in Pichia pastoris, using PLA2, as a fusion partner, J. Mol. Catal. B Enzym. 125(2016) 81-87.

[21] J.F. Li, J. Zhang, Z. Zhang, H.W. Ma, J.X. Zhang, S.Q. Zhang, Production of bioactive human beta-defensin-4 in Escherichia coli, using SUMO fusion partner, Protein J. 29(5) (2010) 314-319.

[22] Y. Liu, L. Ren, L. Ge, Q. Cui, X. Cao, Y. Hou, F. Bai, G. Bai, A strategy for fusion expression and preparation of functional glucagon-like peptide-1(GLP-1) analogue by introducing an enterokinase cleavage site, Biotechnol. Lett. 36(8) (2014) 1675-1680.

[23] R.J. Butkowski, M.E. Noelken, B.G. Hudson, Estimation of the size of collagenous proteins by electrophoresis and gel chromatography, Methods Enzymol. 82(1982) 410-423.

[24] R. Komsa-Penkova, R. Koynova, G. Kostov, B.G. Tenchov, Thermal stability of calf skin collagen type I in salt solutions, BBA-Protein Struct M. 1297(2) (1996) 171-181.

[25] D. Brandenburg, History and diagnostic significance of C-peptide, Exp. Diabetes Res. 2008(8) (2008) 576862.

[26] D.F. Steiner, The proinsulin C-peptide-A multirole model, Exp Diabesity Res. 5(1) (2004) 7-14.

Stability improvement of human collagen α1(I) chain using insulin as a fusion partner

Stability improvement of human collagen α1(I) chain using insulin as a fusion partner

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 5

编辑推荐

Metrics

本文评价

[1]	Dahai Jiang, Zhidi Min, Jing Leng, Huanqing Niu, Yong Chen, Dong Liu, Chenjie Zhu, Ming Li, Wei Zhuang, Hanjie Ying. Characterization of two halophilic adenylate cyclases from Thermobifida halotolerans and Haloactinopolyspora alba[J]. 中国化学工程学报, 2023, 53(1): 56-62.
[2]	Saboura Ashkevarian, Jalil Badraghi, Fatemeh Mamashli, Behdad Delavari, Ali Akbar Saboury. Covalent immobilization and characterization of Rhizopus oryzae lipase on core-shell cobalt ferrite nanoparticles for biodiesel production[J]. 中国化学工程学报, 2021, 37(9): 128-136.
[3]	Qinghong Shi, Yan Sun. Protein A-based ligands for affinity chromatography of antibodies[J]. 中国化学工程学报, 2021, 29(2): 194-203.
[4]	高敏杰, 史仲平. Process Control and Optimization for Heterologous Protein Production by Methylotrophic Pichia pastoris[J]. Chinese Journal of Chemical Engineering, 2013, 21(2): 216-226.
[5]	周冉, 李淑芬, 张大成. Combination of Supercritical Fluid Extraction with Ultrasonic Extraction for Obtaining Sex Hormones and IGF-1 from Antler Velvet[J]. Chinese Journal of Chemical Engineering, 2009, 17(3): 373-380.