[Construction of combined site-directed random mutation libraries of recombinant human lymphotoxin].
To construct the combined site-directed random mutation library of recombinant human Lymphotoxin (rhLT) for in vitro molecular evolution study, and to study the structure and function relationship. The random point mutations at the sites of 46,106 and 130 were individually generated by overlap PCR amplification with the random nucleotide primers. The three point mutations were combined and cloned into pMD-18T vector to construct the combined mutation library. DNA sequencing was used to evaluate the diversity and randomness of the mutation sites. The combined mutation library was re-engineered, inserted in prokaryotic expression vector pBV220, transformed and expressed in Escherichia coli strain DH5alpha. The biological activity of some of the mutants was tested in 1929 mouse fibroblast cells. As much as 1.5 x 10(5) clones were obtained, which represents 4.5 times of the complete mutation libraries at 99% confidence. Sequencing 50 clones revealed no obvious bias in the nucleotide and amino acid mutations at the sites. Among the 30 expressed samples underwent for the bioassay, 70% (21 samples) were inactive, 23.3% (7 samples) had lower activity than rhLT, the remaining 6.7% (2 samples) had higher activity than rhLT. The combined site-directed random mutation library of rhLT has been constructed successfully. In combination with phase display, the library is ready for in vitro molecular evolution study.
Shen
Yi-Jun
YJ
Department of Microbiology, College of Basic Medical Sciences, Second Military Medical University, Shanghai 200433, China.
Pan
Wei
W
Xu
Rong
R
Pan
Xin
X
Wang
Luo-Chun
LC
Wang
Zheng
Z
Cao
Feng
F
Tan
Jing-Wei
JW
Wu
Jin-Song
JS
Wu
Fang
F
Liu
Yan-Jun
YJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D015723:Gene Library; D006801:Humans; D008233:Lymphotoxin-alpha / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics*
D008233:Lymphotoxin-alpha; D011994:Recombinant Proteins