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작성일 : 22-09-13 11:36

(2022, IF=3.705) Optimization of protein trans-splicing in an inducible plasmid display system for high-throughput screening and selection of soluble proteins

글쓴이 : ycpark

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https://www.sciencedirect.com/science/article/pii/S0141022921001721 [639]

Optimization of protein trans-splicing in an inducible plasmid display system for high-throughput screening and selection of soluble proteins

Jinkyeong Yang, Nayeon Kim, Wonbeom Park, Jihwan Chun, Seungjoo Kim, Jongkyeok Shin, Yong-Cheol Park, Kanghee Han, Sun-Ki Kim and Dae-Hyuk Kweon

Enzyme and Microbial Technology. 153: 109914 (2022.01)

Directed evolution is widely used to optimize protein folding and solubility in cells. Although the screening and selection of desired mutants is an essential step in directed evolution, it generally requires laborious optimization and/or specialized equipment. With a view toward designing a more practical procedure, we previously developed an inducible plasmid display system, in which the intein (auto-processing) and Oct-1 DNA-binding (DBD) domains were used as the protein trans-splicing domain and DNA-binding module, respectively. Specifically, the N-terminal (CfaN) and C-terminal (CfaC) domains of intein were fused to the C-terminal end of the His-tag and the N-terminal end of Oct-1 DBD to generate His6-CfaN and CfaC-Oct-1, respectively. For such a system to be viable, the efficiency of protein trans-splicing without the protein of interest (POI) should be maximized, such that the probability of occurrence is solely dependent on the solubility of the POI. To this end, we initially prevented the degradation of l-arabinose (the inducer of the PBAD promoter) by employing an Escherichia coli host strain deficient in the metabolism of l-arabinose. Given that a low expression of His6-CfaN, compared with that of CfaC-Oct-1, was found to be conducive to the generation to a soluble product of the protein trans-splicing event, we designed the expression of His6-CfaN and CfaC-Oct-1 to be inducible from the PBAD and PT7 promoters, respectively. The optimized system thus obtained enabled in vitro selection of the plasmid–protein complex with high yield. We believe that the inducible plasmid display system developed in this study would be applicable to high-throughput screening and/or selection of protein variants with enhanced solubility.

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