前导肽介导的SprD蛋白体外折叠研究

Study on Folding of SprD Protein Mediated by Pro-peptide in vitro

为提高以前体形式合成的蛋白质折叠与成熟效率,以枯草杆菌蛋白酶SprD为研究对象,通过蛋白重组表达、氨基酸定点突变、包涵体纯化结合体外复性、酶催化性能测定等方法比较各前体蛋白折叠成熟情况。结果表明,野生型前导肽介导的SprD前体蛋白可以在230 min内完成成熟,时间较长;成熟肽序列中E112A、S221A及S221C等突变导致相应前体蛋白成熟时间延长或成熟失败;而前导肽串联表达和切割位点酪氨酸缺失等措施可以将蛋白成熟时间缩短至80~160 min,成熟效率提高0.5~2.0倍,且成熟酶催化能力不受影响。前导肽序列中单个氨基酸位点改变对蛋白成熟影响较小,但对酶催化能力提升有一定帮助。因此,前导肽介导的蛋白成熟与酶催化能力的改变是2个相对独立的过程,前导肽表达方式与切割位点变化是促进蛋白体外成熟的较优方案。研究结果为加速蛋白成熟与蛋白改造提供了可参考方法。

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 E112A,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.