Comparison of approaches for site-directed saturation mutagenesis of anarylsulfatase / 西安交通大学学报(医学版)

ABSTRACT

Objective: To compare the overall efficiency and cost of the construction and screening of libraries of Pseudomonas aeruginosa arylsulfatase (PAAS) through site-directed saturation mutagenesis with random primers (NNN) and precise primers for PCR. Methods: Site-directed mutagenesis libraries were constructed using the random primers (NNN) of the selected site, an equal-mole mixture of precise primers and a purified precise primer each time for the amplification of the fusion vector of Escherichia coli alkaline phosphatase (ECAP) and PAAS through PCR. The libraries were screened in a high-throughput mode through the assay of activity ratios of PAAS/mutants to ECAP after alkaline lysis of host cells. Three approaches for site-directed saturation mutagenesis were compared for their number of monoclones screened, the number of their expected mutants discovered, overall time consumed, overall cost and other pertinent factors. Results: The site-directed saturation mutagenesis of M72 with random primers for PCR resulted in only 10 among 20 expected mutants after the screening of over 600 monoclones, besides obvious codon bias. In contrast, no obvious codon bias was observed and there were already 18 among 20 expected mutants after the screening of less than 150 monoclones for site-directed saturation mutagenesis of G138 with similar random primers. The site-directed saturation mutagenesis of M72 with an equal-mole mixture of precise primers yielded all of the 19 expected mutants after the screening of less than 190 monoclones; the site-directed saturation mutagenesis of M72 with the purified precise primers for PCR one-by-one gave all of the 19 expected mutants after the screening of just 2 monoclones for every expected mutant. The use of the purified precise primers for PCR one-by-one was thus more favorable for the construction of libraries of site-directed saturation mutagenesis for the minimum number of monoclones screened, the least overall time and cost. In comparison to the use of the purified precise primers for PCR one-by-one, the use of random primers or the equal-mole mixture of precise primers for PCR to generate the libraries tolerated much greater overall cost and longer time. Conclusion: The generation and screening of the site-directed saturation mutagenesis libraries with precise primers for PCR one-by-one were more practical in elucidating the sequence-activity relationship while the use of equal-mole mixture of precise primers for PCR was preferable for the screening of positive mutants during site-directed saturation mutagenesis.