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

doi:10.1016/j.cjche.2018.04.008

Chin.J.Chem.Eng. ›› 2018, Vol. 26 ›› Issue (12): 2607-2614.DOI: 10.1016/j.cjche.2018.04.008

• Biotechnology and Bioengineering • Previous Articles Next Articles

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:2019-01-09 Published:2018-12-28
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).

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

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).

Abstract

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

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

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]. Chin.J.Chem.Eng., 2018, 26(12): 2607-2614.

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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

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