Denèfle P, Kovarik S, Guitton J D, Cartwright T, Mayaux J F
Génética SA, Joinville le Pont, France.
Gene. 1987;56(1):61-70. doi: 10.1016/0378-1119(87)90158-2.
A synthetic gene coding for human angiogenin was synthesized by solid support phosphoramidite chemistry as eight long oligodeoxynucleotides which were subsequently assembled and cloned in Escherichia coli. The gene was designed to use codons found in highly expressed E. coli proteins. A pBR322-derived expression vector was constructed containing the E. coli trp promoter, the ribosome-binding site of the bacteriophage lambda cII gene, the angiogenin coding sequence, and the transcription terminator region of the E. coli rrnB operon. Under tryptophan deprivation, angiogenin was strongly expressed in E. coli cells at a yield of 5-10% of total protein. The eukaryotic protein was found to be insoluble but could be easily renatured and purified. The purified angiogenin was demonstrated to be active as an angiogenic factor and exhibited a characteristic RNase activity.
通过固相亚磷酰胺化学合成了编码人血管生成素的合成基因，该基因由八个长的寡脱氧核苷酸组成，随后进行组装并克隆到大肠杆菌中。该基因设计使用在高表达的大肠杆菌蛋白质中发现的密码子。构建了一个源自pBR322的表达载体，其中包含大肠杆菌色氨酸启动子、噬菌体λ cII基因的核糖体结合位点、血管生成素编码序列以及大肠杆菌rrnB操纵子的转录终止区。在色氨酸缺乏的情况下，血管生成素在大肠杆菌细胞中强烈表达，产量占总蛋白的5-10%。发现该真核蛋白不溶，但可以很容易地复性和纯化。纯化的血管生成素被证明具有血管生成因子活性，并表现出特征性的核糖核酸酶活性。
