Klein B K, Hill S R, Devine C S, Rowold E, Smith C E, Galosy S, Olins P O
Monsanto Corporate Research, Monsanto Co., St. Louis, MO 63198.
Biotechnology (N Y). 1991 Sep;9(9):869-72. doi: 10.1038/nbt0991-869.
We have expressed a chimeric protein, comprising the LamB secretion signal sequence fused to mature bovine somatotropin (bST), in Escherichia coli. Plasmid constructs with the recA promoter showed significant protein accumulation prior to induction and cell lysis occurred after induction. In contrast, the lacUV5 promoter was tightly regulated. With the lacUV5 promoter, temperature and inducer concentration had significant effects on the total amount of recombinant protein produced and the fraction processed to mature bST. Quantitation of bST from shake flask cultures showed that 1-2 micrograms/ml/OD550 could be released from the periplasm by osmotic shock. N-terminal sequence analysis of the purified protein indicated that the majority of the secreted bST was correctly processed. The bST present in the osmotic shock fraction was judged to be correctly folded by comigration with oxidized methionyl-bST standard on a non-reducing polyacrylamide gel and activity in a bovine liver radioreceptor assay. These results provide a rapid method to produce bST for use in structure-function studies.
我们在大肠杆菌中表达了一种嵌合蛋白，该蛋白由与成熟牛生长激素（bST）融合的LamB分泌信号序列组成。带有recA启动子的质粒构建体在诱导前显示出显著的蛋白质积累，诱导后发生细胞裂解。相比之下，lacUV5启动子受到严格调控。使用lacUV5启动子，温度和诱导剂浓度对产生的重组蛋白总量以及加工成成熟bST的比例有显著影响。摇瓶培养物中bST的定量分析表明，通过渗透休克可从周质中释放出1-2微克/毫升/OD550。纯化蛋白的N端序列分析表明，大多数分泌的bST被正确加工。通过在非还原聚丙烯酰胺凝胶上与氧化甲硫氨酰-bST标准品共迁移以及在牛肝放射受体测定中的活性，判断渗透休克组分中存在的bST折叠正确。这些结果提供了一种快速生产用于结构-功能研究的bST的方法。
