Solid-Phase Cell-Free Protein Synthesis to Improve Protein Foldability

Abstract

Proteins are the most abundant molecules in biology which control virtually every biological process on which our lives depend. Therefore, understanding how newly synthesized proteins folds into the correct native structure and achieve their biologically functional states inside the cell is of paramount importance. Most of what is currently known about the process of protein folding has been studied by analyzing proteins outside the cells in a ‘dilute solution’ under in vitro conditions. The pioneering work on the creation of cell-free (in vitro) protein synthesis (CFPS) reported by Nirenberg and Matthaei in 1961 has been a powerful and ever expanding tool for large-scale analysis of proteins [1]. In general, these systems are derived from the crude extract of cells engaged in a high rate of protein synthesis and are consist of all the macromolecular components required for translation of exogenous mRNA which are added separately in the system. The cell-free system offer several advantages over traditional cell-based (in vivo) systems which are specially not good at making exogenous proteins and those which are toxic to the host cell, undergoes rapid proteolytic degradation or forms inclusion bodies. Cell-free system provides the ability to easily manipulate the reaction components and conditions to favor protein synthesis, decreased sensitivity to product toxicity and suitability for miniaturization and high-throughput applications. With these advantages, there is continuous increasing interest in CFPS system among biotechnologists, molecular biologists and medical or pharmacologists. However, CFPS systems rely on the correct folding of the expressed polypeptide chain into a fully functional three-dimensional protein. Thus ‘foldability’ of expressed protein in a cell-free system is one of the most challenging conundrums of CFPS science.