In vivo modification of native carrier protein domains.
Carrier proteins are central to the biosynthesis of primary and secondary metabolites in all organisms. Here we describe metabolic labeling and manipulation of native acyl carrier proteins in both type I and II fatty acid synthases. By utilizing natural promiscuity in the CoA biosynthetic pathway in combination with synthetic pantetheine analogues, we demonstrate metabolic labeling of endogenous carrier proteins with reporter tags in Gram-positive and Gram-negative bacteria and in a human carcinoma cell line. The highly specific nature of the post-translational modification that was utilized for tagging allows for simple visualization of labeled carrier proteins, either by direct fluorescence imaging or after chemical conjugation to a fluorescent reporter. In addition, we demonstrate the utility of this approach for the isolation and enrichment of carrier proteins by affinity purification. Finally, we use these techniques to identify a carrier protein from an unsequenced organism, a finding that validates this proteomic approach to natural product biosynthetic enzyme discovery.
Mercer
Andrew C
AC
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, USA.
Meier
Jordan L
JL
Torpey
Justin W
JW
Burkart
Michael D
MD
D000213:Acyl Carrier Protein / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D001419:Bacteria / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D045744:Cell Line, Tumor; D002470:Cell Survival; D005227:Fatty Acids / Q000096:biosynthesis; D055785:Gene Knockdown Techniques; D006801:Humans; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D040901:Proteomics; D017422:Sequence Analysis, DNA; D013194:Staining and Labeling; D019014:fas Receptor / Q000378:metabolism
D000213:Acyl Carrier Protein; D000345:Affinity Labels; D001426:Bacterial Proteins; C491901:FAS protein, human; D005227:Fatty Acids; D019014:fas Receptor