Protein folding studies in vivo with a bacteriophage T4 expression-packaging-processing vector that delivers encapsidated fusion proteins into bacteria.
A cloned phage T4 gene which expresses the nonessential capsid scaffold protein IPIII was modified to permit construction and packaging of protein fusions within the capsid. IPIII deletion phage packaged IPIII-beta-galactosidase, IPIII-beta-globin, and IPIII-beta-globin-beta-galactosidase fusion proteins; the latter protein fusion was specifically processed by the T4 gene 21 head morphogenetic proteinase in vivo at a consensus leu(ile)-P1-glu* cleavage site to regenerate beta-galactosidase. Phage inject IPIII-beta-galactosidase protein into bacteria, but less activity is recovered in infections of Escherichia coli dnaK or groEL mutants, suggesting that these host molecular chaperones are required for beta-galactosidase intracellular folding. This expression-packaging-processing (EPP) vector directs protein fusions into capsids for easy detection and purification and permits study of protein delivery and folding in bacteria.
Hong
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YR
Department of Biological Chemistry, University of Maryland Medical School, Baltimore 21201.
Black
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LW
D017122:Bacteriophage T4 / Q000235:genetics*; D001483:Base Sequence; D001692:Biological Transport; D002213:Capsid / Q000235:genetics / Q000378:metabolism; D036022:Capsid Proteins; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D005914:Globins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D036022:Capsid Proteins; D011993:Recombinant Fusion Proteins; C081364:internal protein III, bacteriophage T4; D005914:Globins; D001616:beta-Galactosidase; D010450:Endopeptidases; C081375:gp21 morphogenetic proteinase, phage T4