人转化生长因子β1在大肠杆菌中的分泌表达

Secretion Expression of Human Transforming Growth Factor β1 in E.coli

为了研究双体形式生长因子的原核基因工程，尝试了人转化生长因子β１（ｈＴＧＦβ１）基因的分泌表达．通过缺失突变，构建了能表达具有天然一级结构的ｈＴＧＦβ１单体蛋白的周质分泌表达质粒．采用双顺反子表达系统，使ＴＧＦβ１在周质中获得了高效可溶性表达．研究了改善转运通路对重组蛋白分泌表达的影响，发现共表达σ３２基因和ｄｓｂＡ基因，可促进周质中ＴＧＦβ１双体分子的形成；而共表达ｓｅｃＥ／Ｙ基因对ＴＧＦβ１的分泌表达则没有明显影响．通过共表达ｋｉｌ基因，使ＴＧＦβ１在胞外培养基中获分泌表达，并在胞外折叠。

TGF β1(transforming growth factor β1),a 25 kD homodimer,has widespread biological activities and profound clinical application potential.Human TGF β1 was chosen as a model to study the prokaryotic expression of dimeric growth factors.Secretion expression is reported here as another strategy besides cytoplasmic expression.After the linker region between ompA signal sequence and TGF β1 mature peptide gene was deleted,an expression plasmid which could secret human TGF β1 monomer protein with the natural primary structure into the periplasm of E.coli was constructed.In a dicistron expression system,TGF β1 attained high level and soluble expression in the periplasm.The effect of optimizing the translocation pathway of recombinant protein was studied.It was found that coexpression of σ 32 gene and dsbA gene,could drive the formation of TGF β1 dimer in the periplasm,while coexpression of secE/Y gene had no significant effect on TGF β1 secretion.When kil gene was coexpressed,TGF β1 was secreted into the culture medium,folded and assembled to form the biologically active dimer extracellularly.