【作者】 种慧敏；

【导师】 廖飞；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2017， 硕士

【摘要】 起始酶及其突变体作为目标酶与标签酶融合表达,检测在细胞裂解液中目标酶与标签酶活力并计算其活力比值用于比较是更高效可靠的酶突变体高通量(HTP)筛选策略。适合的标签酶应满足:多肽链一端位于酶活性中心以外从而便于与目标酶融合表达,其酶活性在未转化宿主细胞裂解液中很低故本底很低,融合后对目标酶活力几乎没有影响或对起始酶及其突变体活力产生相同比例的影响;适用的目标酶要求其多肽链一端在活力中心之外以便与标签酶融合表达。碱性磷酸酶和芳香硫酸酯酶有重要应用,且二者的活性能在微孔板中单通道同步测定。本文以大肠杆菌碱性磷酸酶(ECAP)为标签酶置于融合蛋白N端,以铜绿假单胞菌芳香硫酸酯酶(PAAS)为目标酶且置于融合蛋白C端,中间以中性柔性短肽连接。于PAAS M72位进行定点饱和突变建立一个小规模突变体库,用碱裂解方法在微孔板中裂解细胞制样,用酶标仪检测两酶活力并计算PAAS及其突变体与ECAP的活性比值。PAAS及其三个突变体在融合表达时都出现了降解的蛋白片段,但在细胞裂解液中,PAAS及其突变体与ECAP活力比值的相对值与未融合酶/突变体纯化后比活的相对值成正比,这种比较活力比值的方法能够更好地识别弱阳性突变体,即使非融合酶/突变体之间纯化后比活性比值接近1.50时,比较融合酶中两种酶活性相对值识别阳性突变体时,ROC分析所得曲线下面积AUC也能高达0.995,如果仅比较其细胞裂解液中的酶活性则ROC分析的AUC小于0.83。此方法可以高通量筛选突变体库中高活力的突变体、快速确定目标酶的活性序列关系。这种新的方法只需要寻找对于目标酶来说适合的标签酶,此筛选策略在高通量筛选方面具有巨大潜力。更多还原

【Abstract】 A new strategy was proposed for highthroughput(HTP)screening of mutant libraries of a starting enzyme through fusion expression of enzyme/mutants with an enzyme tag to compare their activity ratios to the tag in cell lysates.A suitable enzyme tag should have one terminus nonessential for catalysis,negligible activities in lysates of non-transformed cells and negligible/consistent impacts on activities of the fused partners;an applicable starting enzyme should exhibit its action independent of one terminus.In an experimental model,Escherichia coli alkaline phosphatase(ECAP)as the tag was fused to the N-terminus of Pseudomonas Aeruginosa arylsulfatase(PAAS)through a neutral flexible linker;a small library of PAAS was generated through saturated mutagenesis at M72;cell lysates were prepared by alkaline lysis in microplate wells;enzymatic activities were measured with a microplate reader to derive activity ratios of PAAS/mutants to ECAP.The fused forms of PAAS and mutants in cell lysates tolerated similar fragmentations,but gave activity ratios proportional to specific activities of their purified non-fused counterparts;the comparison of their activity ratios to ECAP in cell lysates gave better recognition of weakly-positive mutants.As for a pair of nonfused PAAS/mutants bearing a ratio of specific activity of about 1.50 after purification,the comparison of their activity ratios to ECAP in their fused forms in cell lysates still gave the area-under-curve(AUC)of about 0.995 for receiver-operator-characteristic(ROC)analysis to recognize the positive one.Meanwhile,the comparison of their activities in cell lysates for the recognition of the positive one gave AUC below 0.83 for ROC analysis.HTP screening of the small library easily revealed positive mutants and sequence-activity relationship of PAAS.With a suitable enzyme tag to an applicable enzyme,this new strategy was promising for HTP screening of mutant libraries.更多还原