EXPRESSION OF POKEWEED (PHYTOLACCA-AMERICANA) ANTIVIRAL PROTEIN CDNA IN ESCHERICHIA-COLI AND ITS ANTIVIRAL ACTIVITY

Two DNA constructs encoding the pokeweed antiviral protein (PAP), one encoding PAP and its signal peptide and the other encoding PAP, were made using polymerase chain reactions (PCR), inserted into the expression vector pKK233-2 and transformed into Escherichia coli JM109 cells. The transformants containing the gene encoding PAP with its signal peptide were able to grow and produce the PAP protein. Two protein bands with molecular weights of 30 kDa and 33 kDa, corresponding to PAP without and with its signal peptide, were detected using monospecific PAP antiserum in Western blots. This suggested that the signal peptide of PAP could be recognized in E. coli but not as efficiently as in pokeweed plant cells. The concentration of PAP expressed in E. coli was low, with only 0·37 mg of PAP produced from II of bacterial culture in LB medium, constituting 0·13% of the total bacterial protein. A slightly higher yield of PAP, 0·9 mg PAP I-1of culture which accounts for 0·16% of total protein, was achieved by culturing in M9 medium. Purified PAP depurinated the rRNA of E. coli as well as yeast and plants and probably inactivates the ribosomes in the same way as other ribosome-inactivating proteins (RIPs). This strong inhibition of E. coli ribosomes may explain the difficulty of expressing the PAP gene in E. coli. The PAP produced in E. coli was able to inhibit infection of tobacco by TMV. © 1993 Academic Press, Inc.