Ptitsyn L R, Al'tman I B
RF GosNII GENETIKA State Research Center, Moscow, Russia.
Bioorg Khim. 1999 Dec;25(12):923-9.
An expression plasmid pPTK-hEGF2 was constructed to provide for the extracellular production of recombinant human epidermal growth factor by the Escherichia coli cells. The plasmid contained two expression cassettes, one of which carried a tandem of the fused genes ompF-hegf under the control of the tac promoter, ensuring regulated secretion of hEGF into the E. coli periplasm, and another one contained the kil gene from the ColE1 plasmid under the control of lac promoter. The regulated low-level biosynthesis of Kil protein increased the permeability of E. coli outer membrane for periplasmic proteins. This enabled the recombinant proteins secreted into the cell periplasm to outflow into the cultural medium. As a result, the E. coli strains that harboured this plasmid construct produced effectively the recombinant hEGF into the cultural medium. The yields of hEGF produced by the nTG1(pPTK-hEGF2) and HB101(pPTK-hEGF2) strains reached 25 and 30 mg/l of cell culture after 14 and 18 h of cultivation, respectively. The hEGF preparation isolated possessed biological activity both in vivo and in vitro.
构建了表达质粒pPTK-hEGF2，用于通过大肠杆菌细胞在细胞外产生重组人表皮生长因子。该质粒包含两个表达盒，其中一个在tac启动子的控制下携带融合基因ompF-hegf的串联体，确保hEGF调节分泌到大肠杆菌周质中，另一个在lac启动子的控制下包含来自ColE1质粒的kil基因。Kil蛋白的低水平生物合成调节增加了大肠杆菌外膜对周质蛋白的通透性。这使得分泌到细胞周质中的重组蛋白能够流出到培养基中。结果，携带该质粒构建体的大肠杆菌菌株有效地将重组hEGF产生到培养基中。在培养14小时和18小时后，nTG1(pPTK-hEGF2)和HB101(pPTK-hEGF2)菌株产生的hEGF产量分别达到25毫克/升和30毫克/升细胞培养物。分离得到的hEGF制剂在体内和体外均具有生物活性。
