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Engineering of a Pichia pastoris Expression System for High-Level Secretion of HSA/GH Fusion Protein

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Abstract

Human serum albumin (HSA) and human growth hormone (hGH) fusion protein [HSA/GH] is a promising long-acting form of GH to treat GH deficiency. This study attempted to engineer a P. pastoris strain for high-level production of HSA/GH to be used in basic research and clinical application. Strains contained two, three, and seven copies of HSA/GH gene were screened by selecting against Zeocin resistance. The results revealed that introducing two to three copies of HSA/GH gene was sufficient to give a significant increase in secretion level, compared with one copy of HSA/GH gene. No significant differences were observed between two to three copies and seven copies. Co-expression with either one copy of exogenous ERO1 or PDI in a strain carrying multicopies of HSA/GH gene led to varying degrees of increase in HSA/GH secretion. The effect of introducing multicopies of PDI was similar to that of one copy of PDI, but introducing excess copies of ERO1 reduced HSA/GH secretion. Simultaneous co-expression with PDI and ERO1 was less effect than either PDI co-expression or ERO1 co-expression. A strain showing higher secretion level was successfully applied to large-scale fermentation with the productivity of 3–4 g/l.

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Abbreviations

HSA:

Human serum albumin

hGH:

Human growth hormone

ER:

Endoplasmic reticulum

PDI:

Protein disulfide isomerase

ERO1:

ER Oxidoreductase 1

ELISA:

Enzyme-linked immunosorbent assay

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Acknowledgment

This work was financially supported by a grant (No. 2010C13006) from the Science and Technology Department of Zhejiang Province, China.

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Authors and Affiliations

  1. Institute of Pharmacology and Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, 310058, People’s Republic of China

    Min Wu, Wenhui Liu, Hongying Sun & Shuqing Chen

  2. Zhejiang Uniongen Biopharm, Huzhou, 313300, People’s Republic of China

    Guanghui Yang, Dengke Yu & Dianhai Lin

Authors
  1. Min Wu
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  2. Wenhui Liu
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  3. Guanghui Yang
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  4. Dengke Yu
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  5. Dianhai Lin
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  6. Hongying Sun
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  7. Shuqing Chen
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Correspondence to Shuqing Chen.

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Wu, M., Liu, W., Yang, G. et al. Engineering of a Pichia pastoris Expression System for High-Level Secretion of HSA/GH Fusion Protein. Appl Biochem Biotechnol 172, 2400–2411 (2014). https://doi.org/10.1007/s12010-013-0688-y

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  • DOI: https://doi.org/10.1007/s12010-013-0688-y

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