Cloning and high expression of hbFGF with a new strategy.
Computer program DNASIS v2.5 was used to help designing the site-directed mutations for optimizing the expression of hbFGF in E. coli. The secondary structure of the translation initiation region (TIR) is a determinant factor for translation initiation rate, meanwhile, codon preference plays an important role, too. According to the two principles, 4 sites in 5' end of hbFGF cDNA were definitely changed, and another 4 sites randomly changed. These mutations will lead to potential variation in the secondary structure of TIR. Then computer program DNASIS v2.5 was utilized to analyse the total 32 TIR sequences resulted from the combination of the 4 randomly mutated sites. Ten sequences with highest free formation energy (delta G0) were chosen for subsequent cloning. By PCR using synthetic primers containing the 8 changed sites described above, ten hbFGF cDNA were amplified and cloned to pET-3c respectively. E. coli strain BL21 (DE3) was transformed and induced to express recombinant hbFGF. Two high-expression clones were obtained by SDS-PAGE and MTT assay, indicating that computer program-aided design for optimizing expression of foreign genes in E. coli is useful.
Song
Zhao-Lei
ZL
Bioengineering Institute of Jinan University, Guangzhou 510632, China.
Pan
Qiu-Hui
QH
Wang
Ju
J
Yang
Yuan
Y
Zhu
Da-Ming
DM
Chen
Xiao-Jia
XJ
Liu
Cui-Lan
CL
Hong
An
A
D003001:Cloning, Molecular / Q000379:methods*; D018387:Codon, Initiator / Q000737:chemistry; D017076:Computer-Aided Design; D004926:Escherichia coli / Q000235:genetics*; D016222:Fibroblast Growth Factor 2 / Q000096:biosynthesis* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D013816:Thermodynamics
D018387:Codon, Initiator; C100666:KCB-1 protein, recombinant; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2