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Improving heterologous expression of porcine follicle-stimulating hormone in Pichia pastoris by integrating molecular strategies and culture condition optimization

  • Applied genetics and molecular biotechnology
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Abstract

Porcine follicle-stimulating hormone (pFSH), comprising α and β subunits, is commonly used to induce superovulation in domestic animals in assisted reproduction technologies; however, the practical application of pFSH is inhibited by the limited efficiency of its production. Recombinant yeast-derived FSH offers a practical alternative; however, the heterologous expression efficiency remains disappointingly low. To improve FSH production in Pichia pastoris, a series of molecular strategies, together with fermentation optimization, were tested in the present study. By comparing clones of the Muts phenotype strain, it was observed that the yield of soluble pFSH increased by approximately 96% in clones of the Mut+ phenotype strain. The protein levels of soluble pFSHβ, which confers biological specificity, increased by approximately 143 and 22% after two kinds of codon optimization strategies, respectively. Moreover, compared with the production of soluble pFSHβ and SUMO-pFSHβ, the production of soluble protein HSA-pFSHβ was significantly improved. Furthermore, the optimum pH and methanol concentration for expressing soluble HSA-pFSH in strain H3-3 were determined as 5.0–6.0 and 1.5–2% in shake-flask, and the yield of soluble HSA-pFSH could reach 40.8 mg/l after purification. In vitro bioactivity assays showed that recombinant HSA-pFSH could efficiently stimulate cAMP synthesis in HEK293 cells expressing porcine FSHR. In conclusion, our results demonstrated that the application of phenotypic selection of aox1 mutants, combined with codon optimization, the choice of fusion partners, and fermentation optimization, considerably increased the yield of pFSH in supernatant of P. pastoris and thus provided a valuable reference for the large-scale recombinant expression of pFSH.

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Acknowledgments

We would like to thank the native English speaking scientists of Elixigen Company (Huntington Beach, California) for editing our manuscript and the staff of Functional Genomic Technology Center Mass Spectrometry Laboratory for mass spectrometry analysis.

Funding

This work was supported by grants from the National Key R&D Program (2017YFD0501901; 2017YFD0501905) and the Earmarked Fund for the Innovative Teams of Beijing Swine Industrialization Research Program.

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Authors and Affiliations

  1. National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, 2 Yuanmingyuan West Road, Beijing, People’s Republic of China

    Gang Luo, JianHui Tian &  Lei An

  2. Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of China

    Huoqing Huang

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  1. Gang Luo
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  2. JianHui Tian
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Luo, G., Tian, J., Huang, H. et al. Improving heterologous expression of porcine follicle-stimulating hormone in Pichia pastoris by integrating molecular strategies and culture condition optimization. Appl Microbiol Biotechnol 102, 8867–8882 (2018). https://doi.org/10.1007/s00253-018-9260-6

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