INCREASING THE EFFICIENCY OF PROTEIN EXPORT IN ESCHERICHIA-COLI

Export of recombinant proteins to the periplasm of Escherichia coli is in many cases preferable to cytoplasmic production. However, when the protein is overexpressed, export efficiency decreases significantly a nd some advantages of the system are lost. This is what happens when a ttempting to produce recombinant human interleukin-6 (hIL-6) as a pre( OmpA) fusion in E. coli. Assuming that the host protein export machine ry becomes overloaded, we have tested the effect of providing the host with additional copies of two key components of that machinery. Suppl ementation with a plasmid bearing prlA4 (sec Y allele) and secE genes increased the ratio of mature to precursor hIL-6 from 1.2 to 10.8. The increase in processing ratio,vas associated with the accumulation of a larger amount of total (mature plus precursor forms) hIL-6. Providin g a plasmid-borne wild-type prlA was ineffective compared to prlA4 all ele. This suggests that the PrlA protein, a component of the transloca tor, recognizes features at the mature portion of secretory substrates independently of those at the signal peptide portion.