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 five-week period of time. Total peptide yields ranged from 0.8 to 16%, which corresponds to 2-70 milligrams 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.

A Multipronged Approach for Compiling a Global Map of Allosteric Regulation in the Apoptotic Caspases

“…We built native peptides derived directly from BIR3 and peptides in which caspase-9 interacting residues were grafted on the stable avian Pancreatic Polypeptide (aPP) scaffold. For peptides made of native amino acids, we developed an expression and purification method for producing long peptides more quickly and with higher fidelity than chemical synthesis (Huber, Olson, & Hardy, 2009). We also incorporated the non-native amino acid aminoisobutyric acid, which is α-methylated, and enforces peptide helicity in some of the designed peptides.…”