Expression of mature pokeweed antiviral protein with or without C-terminal extrapeptide in Escherichia coli as a fusion with maltose-binding protein.
Genomic clones encoding the mature pokeweed antiviral protein with or without C-terminal extrapeptide (PAPMC and PAPM), which have been reported to be highly toxic to E. coli cells, were inserted into the expression vector pMAL-p2. The recombinant PAPs (rPAPMC and rPAPM) were successfully expressed in E. coli at 25 degrees C, being exported to the periplasm as soluble fusions with maltose-binding protein (MBP). The rPAPs were cleaved from MBP by treatment with factor Xa and subsequently purified with final yields of 4.0 mg/liter (rPAPMC) and 5.5 mg/liter (rPAPM). rPAPM was resistant to protease digestion, but the C-terminal extrapeptide appeared to be susceptible and was partially digested by some protease in E. coli. Both rPAPMC and rPAPM were as active as the native PAPM from pokeweed leaves in depurinating rat liver and E. coli ribosomes, while the activities of rPAPMC on both ribosomes were decreased at least 60-fold by fusion with MBP.
Honjo
E
E
Department of Applied Biological Sciences, Saga University, Japan.
Watanabe
K
K
D018528:ATP-Binding Cassette Transporters; D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis; D018744:DNA, Plant / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D018745:Genome, Plant; D058619:Maltose-Binding Proteins; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000096:biosynthesis* / Q000737:chemistry; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D054789:Ribosome Inactivating Proteins, Type 1
D018528:ATP-Binding Cassette Transporters; D000998:Antiviral Agents; D002352:Carrier Proteins; D018076:DNA, Complementary; D018744:DNA, Plant; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C461599:maltose transport system, E coli; D009699:N-Glycosyl Hydrolases; C024275:pokeweed antiviral protein