title: Robust production of a peptide library using methodological synchronization.
abstract: Peptide libraries have proven to be useful in applications such as substrate profiling, drug candidate screening and identifying protein-protein interaction partners. However, issues of fidelity, peptide length, and purity have been encountered when peptide libraries are chemically synthesized. Biochemically produced libraries, on the other hand, circumvent many of these issues due to the fidelity of the protein synthesis machinery. Using thioredoxin as an expression partner, a stably folded peptide scaffold (avian pancreatic polypeptide) and a compatible cleavage site for human rhinovirus 3C protease, we report a method that allows robust expression of a genetically encoded peptide library, which yields peptides of high purity. In addition, we report the use of methodological synchronization, an experimental design created for the production of a library, from initial cloning to peptide characterization, within a 5-week period of time. Total peptide yields ranged from 0.8% to 16%, which corresponds to 2-70 mg of pure peptide. Additionally, no correlation was observed between the ability to be expressed or overall yield of peptide-fusions and the intrinsic chemical characteristics of the peptides, indicating that this system can be used for a wide variety of peptide sequences with a range of chemical characteristics.
authors.affiliation: Chemistry Department, University of Massachusetts at Amherst, Amherst, MA 01003, USA.
authors.forename: Kristen L
authors.initials: KL
authors.lastname: Huber
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Olson
authors.forename: Jeanne A
authors.initials: JA
authors.lastname: Hardy
mesh_terms: D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010191:Pancreatic Polypeptide / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D019151:Peptide Library; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D013879:Thioredoxins; C009725:pancreatic polypeptide, avian; D010191:Pancreatic Polypeptide; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases