Engineering thermal properties of elastin-like polypeptides by incorporation of unnatural amino acids in a cell-free protein synthesis system

Abstract

Based on the central dogma of protein synthesis, traditional methods for protein engineering require that altering protein structure and function must be accompanied by changing the nucleotide sequence of the genes encoding the protein. However, the preparation of a template gene for each individual protein requires a great deal of time and effort, thereby limiting the throughput and scope of studying engineered proteins. In this study, we describe translation-level engineering of proteins using cell-free protein synthesis. Taking advantage of the promiscuity of aminoacyl tRNA synthetases in accepting structurally similar amino acid analogues, unnatural amino acids were introduced into elastin-like polypeptides in place of the corresponding cognate amino acids. Through the incorporation of various analogues and starting from the same gene, the phase transition temperatures of elastin-like polypeptides became tunable. Our results demonstrate the usefulness of cell-free protein synthesis for protein engineering using unnatural amino acids without the need for cloning.

Enhanced Production of Soluble Recombinant Proteins With an In Situ‐Removable Fusion Partner in a Cell‐Free Synthesis System

“…All other reagents were purchased from Sigma (St. Louis, MO). The S12 extract was prepared from the E. coli strain BL21‐Star (DE3) (Invitrogen, Carlsbad, CA) as described previously …”

Cell‐free production and streamlined assay of cytosol‐penetrating antibodies

“…All other chemical reagents were obtained from Sigma–Aldrich and used without further purification. The S12 extract prepared from the Escherichia coli strain BL21 Star (DE3) (Invitrogen, Carlsbad, CA) was used as a source of protein synthesis machinery, as described previously (Catherine et al, ; Oh et al, ). Depending on experiments, BL21 Star (DE3) transformed with the plasmid pTUM4 was used to prepare the S12 extract after being induced to overexpress four periplasmic foldases (DsbA, DsbC, FkpA, and SurA; Schlapschy et al, ).…”

Engineering the Architecture of Elastin‐Like Polypeptides: From Unimers to Hierarchical Self‐Assembly

“…Therefore, the ELP has a lower T t at a higher molecular weight given the same composition and concentration. The transition temperature can be further tuned by incorporating noncanonical or unnatural amino acids as the guest residue . In an extension of this line of enquiry, MacKay et al.…”