High-throughput-screening Of Enzyme Mutants Through Fusion Expression By Two Enzymes

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.