High-throughput screening of enzyme mutants by comparison of their activity ratios to an enzyme tag

• Published in: Analytical biochemistry Vol. 588; p. 113474
• Main Authors: Li, Yaping, Chong, Huimin, Zhang, Xiang, Yang, Xiaolan, Liao, Fei
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-01-2020
• Subjects: Activity ratio; Enzyme tag; Fusion expression; HTP screening; Mutant libraries; SSA; HTP screening; HTP; PAAS; Activity ratio; Fusion expression; ECAP; Enzyme tag; SA; BSA; SDESA; Mutant libraries; ITA; IPTG
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2019.113474

With Escherichia coli alkaline phosphatase (ECAP) as the tag fused to the N-terminus of Pseudomonas Aeruginosa arylsulfatase (PAAS) and its mutants via a flexible linker, the comparison of the activity ratios of an applicable enzyme and its mutants to a suitable enzyme tag in cell lysates of their fused forms was tested for high-throughput (HTP) screening of mutants. After both the induced expression of a fused form and alkaline lysis of the transformed cells in microplate wells, HTP assay of the activities of ECAP and PAAS/mutant was realized via spectrophotometric-dual-enzyme-simultaneous-assay to derive their activity ratio. The successful induced expression of fused forms required ECAP activities higher than 5.3 U/L in cell lysates. Of three representative fused PAAS/mutants in cell lysates, there were similar proteolytic fragments and the comparison of their activity ratios greatly enhanced the recognition of weakly positive mutants. After saturation mutagenesis at M72 of the fused PAAS, the activity ratios of PAAS/mutants to ECAP in cell lysates of their fused forms were proportional to specific activities of their non-fused counterparts in cell lysates by an immunoturbidimetric assay. Therefore, the proposed strategy was absorbing for both HTP screening of mutants and HTP elucidation of sequence-activity relationship of applicable enzymes.
With applicable enzyme/mutants fused to an enzyme tag, the activity ratios of their fused forms in cell lysates linearly responded to specific activities of their non-fused counterparts in cell lysates, supporting an absorbing strategy for both HTP screening of mutants and HTP elucidation of sequence-activity relationship. [Display omitted]
•Escherichia coli alkaline phosphatase was utilized as a fusion enzyme tag.•The tag was fused to Pseudomonas Aeruginosa arylsulfatase (PAAS) and mutants.•The induction of such fused forms and lysis of cells were realized in microplates.•Two enzyme activities in lysates were assayed simultaneously via a microplate reader.•Activity ratios of PAAS/mutants to the tag easily recognize weakly-positive mutants.