Study on Folding of SprD Protein Mediated by Pro-peptide in vitro
To improve the folding and maturation efficiency of proteins that were first synthesized as precursors,the bacterial-derived SprD subtilisin was used as the research object and the folding and maturation of precursors were compared comprehensively by taking recombination expression of proteins,site-directed mutation of amino acids,purification of inclusion bodies,refolding of proteins in vitro and determination of enzyme catalytic abilities many other measures.The resluts showed that SprD precursors took about 230 min to complete the maturation under the mediation of wild-type pro-peptides.Mutations of E1 12A,S221A and S221C in sequences of mature peptide led to the prolongation or the failure of maturation.The tandem expression of pro-peptide and deletion of tyrosine at cleavage site between pro-peptide and mature peptide shortened the maturation time to 80-160 min and increased the maturation efficiency by 0.5-2.0 times,and the catalytic abilities of the corresponding mature enzymes were not affected.Although the single-site mutations in pro-peptide had little effects on maturation,they contributed the improvement of catalytic ability.Therefore,the maturation of proteins mediated by pro-peptides and the alternation of enzyme catalytic ability were 2 relatively independent processes and the expression modes of pro-peptide and the changes of cleavage site were suitable ways to promote protein maturation in vitro.Above results would provide a reference method for accelerating protein maturation and protein modification.
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