Manipulation of carrier proteins in antibiotic biosynthesis.
Engineering biosynthetic pathways into suitable host organisms has become an attractive venue for the design, evaluation, and production of small molecule therapeutics. Polyketide (PK) and nonribosomal peptide (NRP) synthases have been of particular interest due to their modular structure, yet routine cloning and expression of these enzymes remains challenging. Here we describe a method to covalently label carrier proteins from PK and NRP synthases using the enzymatic transfer of a modified coenzyme A analog by a 4'-phosphopantetheinyltransferase. Using this method, carrier proteins can be loaded with single fluorescent or affinity reporters, providing novel entry for protein visualization, Western blot identification, and affinity purification. Application of these methods provides an ideal tool to track and quantify metabolically engineered pathways. Such techniques are valuable to measure protein expression, solubility, activity, and native posttranslational modification events in heterologous systems.
La Clair
James J
JJ
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Foley
Timothy L
TL
Schegg
Tracy R
TR
Regan
Conor M
CM
Burkart
Michael D
MD
D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002846:Chromatography, Affinity; D003065:Coenzyme A / Q000096:biosynthesis; D015195:Drug Design; D005456:Fluorescent Dyes / Q000032:analysis; D010453:Peptide Synthases / Q000737:chemistry*; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational
D000900:Anti-Bacterial Agents; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; D003065:Coenzyme A