Co-expression of glutathione S-transferase with methionine aminopeptidase:a system of producing enriched N-terminal processed proteins in Escherichia coli

We describe here an Escherichia coli expression system that produces recomb inant proteins enriched in the N-terminal processed form, by using glutathi one S-transferase cGSTM1-1 and rGSTT1-1 as models, where c and r refer to c hick and rat respectively. Approximately 90% of the cGSTM1-1 or rGSTT1-1 ov erexpressed in E. coli under the control of a phoA promoter retained the in itiator methionine residue that was absent from the mature isoenzymes isola ted from tissues. The amount of initiator methionine was decreased to 40% o f the expressed cGSTM1-1 when the isoenzyme was co-expressed with an exogen ous methionine aminopeptidase gene under the control of a separate phoA pro moter. The recombinant proteins expressed were mainly methionine aminopepti dase. The yield of cGSTM 1-1 was decreased to 10% of that expressed in the absence of the exogenous methionine aminopeptidase gene. By replacing the p hoA with its natural promoter, the expression of methionine aminopeptidase decreased drastically. The yield of the coexpressed cGSTM1-1 was approx. 60 % of that in the absence of the exogenous methionine aminopeptidase gene; a pprox. 65% of the initiator methionine residues were removed from the enzym e. 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 cG STM-11 was decreased to 14% of the overexpressed isoenzymes, whereas no fur ther improvement could be observed for rGSTT1-1. The initiator methionine r esidue does not affect the enzymic activities of either cGSTM1-1 or rGSTT1- 1. However, the epoxidase activity and the 4-nitrobenzyl chloride-conjugati ng activity of the purified recombinant rCSTT1-1 are markedly higher that t hose reported recently for the same isoenzyme isolated from rat livers.