人酸性成纤维细胞生长因子在大肠杆菌的高效表达

Overexpression of human acidic fibroblast growth factor in Escherichia coli

目的 :探讨人酸性成纤维细胞生长因子 (acidic fibroblast growth factor,a FGF)在大肠杆菌中高效表达的方法及重组人 a FGF的生物学活性。方法 :1通过 PCR法扩增人 a FGF引入点突变 ,对其密码子进行改造 ;2构建 a FGF- p ET- 2 8a重组质粒并在大肠杆菌 BL2 1 (DE3)中表达 ;3重组蛋白的纯化及生物学活性研究。结果 :通过 PCR扩增 ,将设计的核酸水平突变引入到 a FGF DNA中 ,使其自起始密码 ATG后的前 5 0个碱基均为原核细胞偏爱密码子、并保持原氨基酸序列不变 ,成功地实现了重组人 a FGF在大肠杆菌中的高效表达 ,并通过降低培养温度使大肠杆菌中可溶性目的蛋白的含量大幅增加 ,原核表达的 a FGF亦具有天然生物学活性。结论 :通过对 a FGF DNA碱基进行改造 ,可大幅度提高 a FGF在大肠杆菌中的表达量 。

Objective To overexpress human acidic fibroblast growth factor (aFGF) in Escherichia coli (E coli ) and study the biological activities of recombinant aFGF. Methods ①The codon of aFGF was altered by amplifying aFGF DNA and introducing point mutation; ②Human aFGF was recombined with pET 28a and expressed in E coli ; ③Recombinant protein was purified and the biological activities were studied. Results The projected point mutation was introduced into aFGF DNA fragment and the first 50 bp after ATG start codon of aFGF were altered to the partiality codon of E coli by PCR, and the original sequence of amino acid was kept. So the overexpression of recombinant aFGF in E coli was successfully accomplished. The expression volume of soluble aFGF in E coli was greatly increased by lowering culture temperature. Recombinant aFGF protein had biological activities of nature aFGF. Conclusion By altering the bases of aFGF DNA, the expression volume of aFGF in E coli is increased greatly, and the recombinant protein has nature biological activities.