Cloning and high level nonfusion expression of recombinant human basic fibroblast growth factor in Escherichia coli.
OBJECTIVE
To obtain high-level expression of nonfusion recombinant human basic fibroblast growth factor (rhbFGF).


METHODS
hbFGF cDNA was prepared from the total RNA of embryonic brain tissue. As a template, the obtained gene was used to clone nonfusion rhbFGF. New primers were employed to alter the translation initiation region (TIR) and reduce the G+C content through nucleotide change. Using pET-3C as vector, the cloned rhbFGF was expressed in BL21 (DE3).


RESULTS
rhbFGF was expressed in E coli up to 30 % of the total cellular protein. Cation exchange and heparin affinity chromatography were employed to purify the target protein from the supernatant of bacteria lysate. The bioactivity of the purified rhbFGF was identical with the standard bFGF.


CONCLUSIONS
Modification of TIR is an effective means to increase nonfusion expression rate of recombinant proteins, such as rhbFGF, in E coli.
Chen
Xiao-Jia
XJ
Bioengineering Institute of Jinan University, Guangzhou 510632, China. ojds@jnu.edu.cn
Sun
Fen-Yong
FY
Xie
Qiu-Ling
QL
Liao
Mei-De
MD
Zhang
Ling
L
Li
Zhi-Ying
ZY
Hong
An
A
Lin
Jian
J
D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005333:Fetus; D016222:Fibroblast Growth Factor 2 / Q000096:biosynthesis* / Q000235:genetics; D005815:Genetic Code; D006801:Humans; D008969:Molecular Sequence Data; D011506:Proteins / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D018076:DNA, Complementary; D011506:Proteins; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2