Mutant library construction in directed molecular evolution: casting a wider net.
Directed molecular evolution imitates the natural selection process in the laboratory to find mutant proteins with improved properties in the expected aspects by exploring the encoding sequence space. The success of directed molecular evolution experiment depends on the quality of artificially prepared mutant libraries and the availability of convenient high-throughput screening methods. Well-prepared libraries promise the possibility of obtaining desired mutants by screening a library containing a relatively small number of mutants. This article summarizes and reviews the currently available methodologies widely used in directed evolution practices in the hope of providing a general reference for library construction. These methods include error-prone polymerase chain reaction (epPCR), oligonucleotide-based mutagenesis, and genetic recombination exemplified by DNA shuffling and its derivatives. Another designed method is also discussed, in which B-lymphocytes are fooled to mutate nonantibody foreign proteins through somatic hypermutation (SHM).
Wang
Tian-Wen
TW
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, P. R. China.
Zhu
Hu
H
Ma
Xing-Yuan
XY
Zhang
Ting
T
Ma
Yu-Shu
YS
Wei
Dong-Zhi
DZ
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D019020:Directed Molecular Evolution / Q000379:methods* / Q000639:trends; D019143:Evolution, Molecular; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D016296:Mutagenesis; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000639:trends