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Enhanced Expression of Recombinant Elastase in Pichia pastoris through the Substitution of Thr for Ser in Asn-Xaa-Ser Sequons

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Abstract

N-glycosylation usually occurs at the Asn-Xaa-Ser/Thr sequon of glycoproteins in Pichia pastoris, exerting great effects on expression efficiency; however, Asn-Xaa-Thr is more efficiently glycosylated than Asn-Xaa-Ser. In this study, the role of the two sequons in the expression of recombinant elastase (rPAE) was investigated. At N43, N212, and N280 of rPAE, Asn-Xaa-Thr was substituted for the native Asn-Xaa-Ser sequon through site-directed mutagenesis, and the two sequon forms were introduced into rPAE at N36 and N264. As expected, substitution at N36, N43, N212, and N280 enhanced the degree of N-glycosylation. At N212 or N280, substitution increased rPAE production effectively by 43 and 25 %, respectively. In comparison, at N36, N43, and N264, the change inhibited rPAE expression to varying extents; specifically, substitution at N36 resulted in a 31 % decrease, while substitution at N43 or N264 resulted in a decrease of less than 9 %. It is suggested that the effect of the substitution of Asn-Xaa-Thr for Asn-Xaa-Ser on rPAE expression is roughly related to the role of the original Asn-Xaa-Ser sequon. As the conversion of Ser to Thr at N-glycosylation sites through site-directed mutagenesis is easily achieved, it is a feasible means of improving the expression of recombinant proteins in P. pastoris.

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Acknowledgments

This work was supported by Open Project of Jiangsu Key Laboratory for Biomass-based Energy and Enzyme Technology (Grant No. JSBEET1306).

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Authors and Affiliations

  1. Jiangsu Key Laboratory for Eco-Agricultural Biotechnology around Hongze Lake; Jiangsu Key Laboratory for Biomass-based Energy and Enzyme Technology, School of Life Sciences, Huaiyin Normal University, Huaian, 223300, China

    Minghai Han, Xinfeng Wang, Xin Liu, Hui Cao & Yuan Tao

  2. Huaian Institute of Supervision & Inspection on Product Quality, Huaian, 223300, China

    Weixian Wang

  3. Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi, 214122, China

    Xiaobin Yu

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  1. Minghai Han
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  2. Weixian Wang
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  3. Xinfeng Wang
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  4. Xin Liu
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  5. Hui Cao
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  6. Yuan Tao
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  7. Xiaobin Yu
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Correspondence to Minghai Han.

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Han, M., Wang, W., Wang, X. et al. Enhanced Expression of Recombinant Elastase in Pichia pastoris through the Substitution of Thr for Ser in Asn-Xaa-Ser Sequons. Appl Biochem Biotechnol 175, 428–435 (2015). https://doi.org/10.1007/s12010-014-1284-5

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  • DOI: https://doi.org/10.1007/s12010-014-1284-5

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