Characterization of Protein Domain Function via in vitro DNA Shuffling
Kathy Po,
Edward Chan,
Sheng Chen
Affiliations
Kathy Po
Shenzhen Key Lab for Food Biological Safety Control, Food Safety and Technology Research Center, Hong Kong PolyU Shen Zhen Research Institute, Shenzhen, P. R. ChinaState Key Lab of Chirosciences, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong
Edward Chan
Shenzhen Key Lab for Food Biological Safety Control, Food Safety and Technology Research Center, Hong Kong PolyU Shen Zhen Research Institute, Shenzhen, P. R. ChinaState Key Lab of Chirosciences, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong
Sheng Chen
Shenzhen Key Lab for Food Biological Safety Control, Food Safety and Technology Research Center, Hong Kong PolyU Shen Zhen Research Institute, Shenzhen, P. R. ChinaState Key Lab of Chirosciences, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong
We recently investigated the molecular events that drive evolution of the CTX-M-type β-lactamases by DNA shuffling of fragments of the blaCTX-M-14 and blaCTX-M-15 genes. Analysis of a total of 51 hybrid enzymes showed that enzymatic activity could be maintained in most cases, yet the enzymatically active hybrids were found to possess much fewer amino acid substitutions than the few hybrids that became inactive, suggesting that point mutations in the constructs rather than reshuffling of the fragments of the two target genes would more likely cause disruption of CTX-M activity. Certain important residues that played important functional roles in mediating enzyme activity were identified. These findings suggest that DNA shuffling is an effective approach to identify and characterize important functional domains in bacterial proteins.
