Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Modulation of Kex2p Cleavage Site for In Vitro Processing of Recombinant Proteins Produced by Saccharomyces cerevisiae

  • Mi-Jin Kim (Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Se-Lin Park (Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Seung Hwa Kim (Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Hyun-Joo Park (Cellapy Bio Inc.) ;
  • Bong Hyun Sung (Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Jung-Hoon Sohn (Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Jung-Hoon Bae (Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology)
  • Received : 2023.06.09
  • Accepted : 2023.06.26
  • Published : 2023.11.28
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Abstract

Kex2 protease (Kex2p) is a membrane-bound serine protease responsible for the proteolytic maturation of various secretory proteins by cleaving after dibasic residues in the late Golgi network. In this study, we present an application of Kex2p as an alternative endoprotease for the in vitro processing of recombinant fusion proteins produced by the yeast Saccharomyces cerevisiae. The proteins were expressed with a fusion partner connected by a Kex2p cleavage sequence for enhanced expression and easy purification. To avoid in vivo processing of fusion proteins by Kex2p during secretion and to guarantee efficient removal of the fusion partners by in vitro Kex2p processing, P1', P2', P4, and P3 sites of Kex2p cleavage sites were elaborately manipulated. The general use of Kex2p in recombinant protein production was confirmed using several recombinant proteins.

Keywords

Acknowledgement

This work was partly supported by High Value-added Food Technology Development Program funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA)(321026051SB010), Agriculture Science and Technology Development Program funded by the Rural Development Administration of Korea(PJ0149382022), the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (2022M3A9l308236712), and the Research Initiative Program(1711196158) of KRIBB.

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