Expression and secretion of Mirabilis antiviral protein in Escherichia coli and its inhibition of in vitro eukaryotic and prokaryotic protein synthesis.
Mirabilis antiviral protein (MAP), a ribosome-inactivating protein, exhibits inhibitory effects on both plant virus infection and protein synthesis. To study these functions by site-specific mutagenesis, the total synthetic gene of MAP was constructed and expressed in Escherichia coli. However, the growth of the host was inhibited by the products, and the yield of MAP was very low. To improve the system for expressing MAP, an expression vector, pSH7, was constructed. This vector is based on the high copy number plasmid pUC19 and includes PL promoter and temperature-sensitive cI857 repressor. The plasmid also contains the ompA signal sequence and the total synthetic MAP gene. The MAP gene was expressed and its product was secreted into the culture medium after E. coli transformants were cultivated at 30 degrees C and the temperature was raised to 42 degrees C. The secreted MAP was then purified and characterized. This protein was identical to native MAP as determined by its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the amino acid sequence at the NH2 terminus, and its inhibitory effect on in vitro protein synthesis. MAP was found to inhibit the in vitro protein synthesis of rabbit reticulocyte and wheat germ. It further showed an IC50 concentration of approximately 200 nM in an E. coli in vitro translation system in contrast to ricin A-chain, a well known ribosome-inactivating protein.
Habuka
N
N
Life Science Research Laboratory, Japan Tobacco, Inc., Kanagawa.
Akiyama
K
K
Tsuge
H
H
Miyano
M
M
Matsumoto
T
T
Noma
M
M
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009154:Mutation; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis / Q000187:drug effects; D021382:Protein Sorting Signals / Q000235:genetics; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D054789:Ribosome Inactivating Proteins, Type 1; D012276:Ricin / Q000494:pharmacology; D014169:Transformation, Bacterial
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D010940:Plant Proteins; D021382:Protein Sorting Signals; D011500:Protein Synthesis Inhibitors; D054789:Ribosome Inactivating Proteins, Type 1; D012276:Ricin; D009699:N-Glycosyl Hydrolases; C059351:MAP protein, Mirabilis jalapa