High level expression of oryzacystatin in Escherichia coli.

A rice cDNA library of immature seeds has already been constructed, from which colonies carrying oryzacystatin cDNA were isolated. The coding region of the oryzacystatin (a thiol proteinase inhibitor) gene was amplified by the Polymerase Chain Reaction (PCR) and inserted downstream of lambdaPRPL promoter of E. coli thermal-inducible expression vector pBV220. An oryzacystatin expression plasmid pBVC9 was therefore obtained. Shifting the culture temperature of E. coli DH5 alpha (pBVC9) from 30 degrees C to 42 degrees C led to a high level expression of oryzacystatin. The result of SDS-PAGE showed a distinct band of 12.0 kDa which accounts for at least 10% of the total soluble proteins. The inhibitory activity of the total soluble proteins of E. coli DH5 alpha (pBVC9) toward papain was confirmed by using that of E. coli DH5 alpha (pBV220) as a control.