A fusion plasmid for the synthesis of lipopeptide-antigen chimeras in Escherichia coli.
Lipopeptides are potential vaccine candidates with a built-in adjuvant property. To circumvent the present chemical route of synthesis for lipopeptide-antigen conjugates, the lipoprotein property of the pColE2-P9-encoded lysis protein, CelB, was used to create the bacterial fusion plasmid, pKLY3, to produce lipopeptide-antigen chimeras in Escherichia coli. Plasmid pKLY3 is a derivative of pKK233-2 with the origin of replication of the single-stranded DNA phage, fl. Under control of the promoter, ptrc, is the 5' end of the celB gene coding for a lipoprotein signal peptide and the first five amino acids (aa) (CQANY) of the mature lysis protein. As model systems for the synthesis of small and large lipopeptide-antigens, DNA sequences coding for the P2 peptide and E. coli alkaline phosphatase (PhoA) were fused in frame to the region of celB coding for a lipoprotein signal peptide and CQANY. P2 is a 12-aa peptide including a tyrosine phosphorylation site of the epidermal growth factor receptor (EGF-R). Inducible expression of stable lipohexapeptide CQANYV, lipo-CQANY-P2, and lipo-CQANYA-PhoA, was demonstrated. Similar expression was obtained for lipo-CIEGR-P2 and lipo-CIEGRA-PhoA in which IEGR is a cleavage recognition site for the blood coagulation factor, Xa. Like QANY, IEGR is predicted to form a beta-turn structure. The presence of a lipid moiety on the products was confirmed by demonstrating the incorporation of radioactive palmitic acid and inhibition of processing by globomycin. The lipid-modified peptides were also identified by incorporation of radioactive tyrosine, and the nature of the P2 peptide was verified immunologically.(ABSTRACT TRUNCATED AT 250 WORDS)
Rioux
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Genetic Engineering Section, National Research Council of Canada, Montreal, Quebec.
Bergeron
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Lin
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Grothe
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O'Connor-McCourt
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Lau
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PC
D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D000941:Antigens / Q000737:chemistry / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D003087:Colicins / Q000737:chemistry; D066246:ErbB Receptors / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D008074:Lipoproteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics*
D000941:Antigens; D001426:Bacterial Proteins; D003087:Colicins; D008074:Lipoproteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D066246:ErbB Receptors; D000469:Alkaline Phosphatase