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.
Department of Biological Chemistry, University of Maryland Medical School, Baltimore 21201.
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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