重组人碱性成纤维细胞生长因子在大肠杆菌中的克隆及高效非融合表达
Cloning and high level nonfusion expression of recombinant human basic fibroblast growth factor in Escherichia coli.
作者信息
Chen Xiao-Jia, Sun Fen-Yong, Xie Qiu-Ling, Liao Mei-De, Zhang Ling, Li Zhi-Ying, Hong An, Lin Jian
机构信息
Bioengineering Institute of Jinan University, Guangzhou 510632, China.
出版信息
Acta Pharmacol Sin. 2002 Sep;23(9):782-6.
AIM
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.
CONCLUSION
Modification of TIR is an effective means to increase nonfusion expression rate of recombinant proteins, such as rhbFGF, in E coli.
目的
获得非融合重组人碱性成纤维细胞生长因子（rhbFGF）的高水平表达。
方法
从胚胎脑组织的总RNA中制备hbFGF cDNA。以获得的基因为模板，用于克隆非融合rhbFGF。采用新引物改变翻译起始区域（TIR），并通过核苷酸变化降低G+C含量。以pET-3C为载体，将克隆的rhbFGF在BL21（DE3）中表达。
结果
rhbFGF在大肠杆菌中表达量高达细胞总蛋白的30%。采用阳离子交换和肝素亲和层析从细菌裂解物的上清液中纯化目标蛋白。纯化的rhbFGF的生物活性与标准bFGF相同。
结论
TIR的修饰是提高重组蛋白如rhbFGF在大肠杆菌中非融合表达率的有效手段。
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