Hwang D D, Liu L F, Kuan I C, Lin L Y, Tam T C, Tam M F
Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, R.O.C.
Biochem J. 1999 Mar 1;338 ( Pt 2)(Pt 2):335-42.
We describe here an Escherichia coli expression system that produces recombinant proteins enriched in the N-terminal processed form, by using glutathione S-transferase cGSTM1-1 and rGSTT1-1 as models, where c and r refer to chick and rat respectively. Approximately 90% of the cGSTM1-1 or rGSTT1-1 overexpressed in E. coli under the control of a phoA promoter retained the initiator methionine residue that was absent from the mature isoenzymes isolated from tissues. The amount of initiator methionine was decreased to 40% of the expressed cGSTM1-1 when the isoenzyme was co-expressed with an exogenous methionine aminopeptidase gene under the control of a separate phoA promoter. The recombinant proteins expressed were mainly methionine aminopeptidase. The yield of cGSTM1-1 was decreased to 10% of that expressed in the absence of the exogenous methionine aminopeptidase gene. By replacing the phoA with its natural promoter, the expression of methionine aminopeptidase decreased drastically. The yield of the co-expressed cGSTM1-1 was approx. 60% of that in the absence of the exogenous methionine aminopeptidase gene; approx. 65% of the initiator methionine residues were removed from the enzyme. Under similar conditions, N-terminal processing was observed in approx. 70% of the recombinant rGSTT1-1 expressed. By increasing the concentration of phosphate in the growth medium, the amount of initiator methionine on cGSTM1-1 was decreased to 14% of the overexpressed isoenzymes, whereas no further improvement could be observed for rGSTT1-1. The initiator methionine residue does not affect the enzymic activities of either cGSTM1-1 or rGSTT1-1. However, the epoxidase activity and the 4-nitrobenzyl chloride-conjugating activity of the purified recombinant rGSTT1-1 are markedly higher that those reported recently for the same isoenzyme isolated from rat livers.
我们在此描述一种大肠杆菌表达系统，该系统通过使用谷胱甘肽S-转移酶cGSTM1-1和rGSTT1-1作为模型来产生富含N端加工形式的重组蛋白，其中c和r分别指鸡和大鼠。在phoA启动子控制下在大肠杆菌中过表达的cGSTM1-1或rGSTT1-1中，约90%保留了从组织中分离的成熟同工酶中不存在的起始甲硫氨酸残基。当该同工酶与在外源phoA启动子控制下的外源性甲硫氨酸氨肽酶基因共表达时，起始甲硫氨酸的量降至所表达的cGSTM1-1的40%。所表达的重组蛋白主要是甲硫氨酸氨肽酶。cGSTM1-1的产量降至无外源性甲硫氨酸氨肽酶基因时所表达产量的10%。通过用其天然启动子替换phoA，甲硫氨酸氨肽酶的表达急剧下降。共表达的cGSTM1-1的产量约为无外源性甲硫氨酸氨肽酶基因时产量的60%；约65%的起始甲硫氨酸残基从该酶中被去除。在相似条件下，在所表达的约70%的重组rGSTT1-1中观察到N端加工。通过增加生长培养基中磷酸盐的浓度，cGSTM1-1上起始甲硫氨酸的量降至过表达同工酶的14%，而对于rGSTT1-1未观察到进一步改善。起始甲硫氨酸残基不影响cGSTM1-1或rGSTT1-1的酶活性。然而，纯化的重组rGSTT1-1的环氧化酶活性和4-硝基苄基氯结合活性明显高于最近报道的从大鼠肝脏分离的同一同工酶。
