Improving the affinity of an anti-TNF? scFv by error prone PCR and staggered extention process(StEP) / 中国免疫学杂志

Objective:To improve the affinity of an anti TNF? scFv.Methods:Starting from an anti TNF? scFv gene a mutant phage antibody library was generated by error prone PCR.Affinity improved clones were selected and subjected to staggered extension process to shuffle the mutated sites.Mutants with further improved affinity were selected by bio panning.Affinity was judged by dot blot ELISA and thiocyanate elusion ELISA.Results:Seven affinity improved mutants were obtained from library constructed by error prone PCR.By StEP mediated shuffling of these 7 clones and via bio panning,mutants with further improved affinity were obtained.Conclusion:Combination of error prone PCR and StEP could be used to improve the affinity of antibodies. [

The condition for error-prone PCR induced mutation / 第二军医大学学报

Objective: To evaluate the effectiveness of random mutagenesis via mutator E.coli strain and error prone PCR. Methods: A ScFv containing phagemid was transformed into mutator strain XL1 Red and subjected to 7 overnight growth phases with 1/100 dilution of each phase. DNA was extracted and sequenced. The ScFv gene was also subjected to PCR mutagenesis. Mutation effects of Mg 2+ concentration, alteration of individual dNTP amounts and addition of dITP were investigated. Results: The mutation rate of 7 growth phases (about 50 cell cycles) in XL1 Red was less than 0.1%. In error prone PCR, higher concentration of Mg 2+ increased the mutation rate. Increased content of dTTP and dCTP had better effect than that of dATP and dGTP. Addition of dITP plus low concentration dATP or dGTP caused lower mutation rate. More than 2% mutation could be reached by 2 rounds PCR containing 5 mmol/L MgCl 2, 0.5 mmol/L MnCl 2, 1 mmol/L dTTP and 1 mmol/L dCTP. But the mutation showed obvious base bias with most substitutions at A/T positions and a prevalence of transition over transversion. Conclusion: Random mutagenesis in mutator strain has too low mutation rate for antibody affinity maturation. A more than 2% mutation rate can be obtained by error prone PCR, which is suitable for constructing mutated antibody libraries, but the base bias of error prone PCR should be considered.