Hayase N, Ishiyama A, Niwano M
Biotechnology Research Laboratory, Kobe Steel Ltd., Hyogo, Japan.
Appl Environ Microbiol. 1994 Sep;60(9):3336-42. doi: 10.1128/aem.60.9.3336-3342.1994.
We constructed the broad-host-range human epidermal growth factor (EGF) secretion plasmid pKSEGF2 by inserting the Escherichia coli tac promoter, the signal sequence of Pseudomonas stutzeri amylase, and the synthesized EGF gene into the broad-host-range vector pKT230. E. coli JM109 carrying pKSEGF2 secreted EGF into the periplasm and the culture medium under the control of the tac promoter. Pseudomonas aeruginosa PAO1161 carrying pKSEGF2 and Pseudomonas putida AC10 carrying pKSEGF2 secreted EGF into the culture medium constitutively. Four hydrogen-utilizing bacteria, Pseudomonas pseudoflava, Alcaligenes eutrophus, Alcaligenes paradoxus, and Paracoccus denitrificans, were transconjugated with pKSEGF2 from eight hydrogen-utilizing bacteria tested. In these transconjugated hydrogen-utilizing bacteria, P. pseudoflava carrying pKSEGF2 grew autotrophically and secreted EGF, confirmed by Western blot (immunoblot) analysis, into the culture medium constitutively.
我们通过将大肠杆菌tac启动子、施氏假单胞菌淀粉酶的信号序列和合成的表皮生长因子（EGF）基因插入广宿主范围载体pKT230，构建了广宿主范围的人表皮生长因子分泌质粒pKSEGF2。携带pKSEGF2的大肠杆菌JM109在tac启动子的控制下将EGF分泌到周质和培养基中。携带pKSEGF2的铜绿假单胞菌PAO1161和携带pKSEGF2的恶臭假单胞菌AC10组成型地将EGF分泌到培养基中。从测试的8种利用氢气的细菌中，用pKSEGF2对4种利用氢气的细菌——假黄色假单胞菌、真养产碱菌、奇异产碱菌和反硝化副球菌进行了转接合。在这些转接合的利用氢气的细菌中，携带pKSEGF2的假黄色假单胞菌自养生长，并通过蛋白质免疫印迹（免疫印迹）分析证实，组成型地将EGF分泌到培养基中。
