Chang-Gil Park

Providing an Oxidizing Environment for the Cell‐Free Expression of Disulfide‐Containing Proteins by Exhausting the Reducing Activity of Escherichia coli S30 Extract

We developed a novel method of producing proteins containing multiple disulfide bonds in a cell‐free protein synthesis system. To provide an optimized redox potential during the synthesis of truncated plasminogen activator (rPA), we pretreated the E. coli S30 extract with an excess amount of oxidized glutathione based on the anticipation that the reducing potential of the S30 extract would be exhausted through the reduction of the oxidized glutathione molecules. As expected, it was found that the reducing activity of the S30 extract was remarkably decreased through the pretreatment, and active rPA was produced when the pretreated S30 extract was used after removing the residual glutathione molecules. In particular, compared to the method involving the iodoacetamide treatment of S30 extract, the present protocol was effective in producing active rPA during the batch reaction of cell‐free protein synthesis.

Expression of functional Candida antarctica lipase B in a cell-free protein synthesis system derived from Escherichia coli.

This article reports the cell‐free expression of functional Lipase B from Candida antarctica (CalB) in an Escherichia coli extract. Although most of the cell‐free synthesized CalB was insoluble under conventional reaction conditions, the combined use of molecular chaperones led to the soluble expression of CalB. In addition, the functional enzyme was generated by applying the optimal redox potential. When examined using p‐nitrophenyl palmitate as a substrate, the specific activity of the cell‐free synthesized CalB was higher than that of the reference protein produced in Pichia pastoris. These results highlight the potential of cell‐free protein synthesis technology as a powerful platform for the rapid expression, screening and analysis of industrially important enzymes.

Cell-free synthesis and multifold screening of Candida antarctica lipase B (CalB) variants after combinatorial mutagenesis of hot spots

We have developed a strategy for rapid and combinatorial optimization of the hot spot residues of enzymes. After combinatorial randomization of target locations in the Candida antarctica lipase B (CalB) gene, the individual variant genes isolated in the E.coli cells were expressed in the cell‐free protein synthesis system to analyze different parameters of the resulting CalB variants. The enzymatic assays for the hydrolysis of para‐nitrophenyl‐ester (pNP‐ester) and triglyceride, synthesis of wax ester, and thermal stability of the variant enzymes were carried out simultaneously in 96‐well microtiter plates. From the 1,000 variant genes tested in each assay, we were able to identify a series of the variant enzymes having markedly improved hydrolytic, synthetic activity, or thermal stability. The improved traits of the cell‐free selected CalB variants were well reproduced when the corresponding genes were expressed in Pichia pastoris. Therefore, we expect that the proposed strategy of cell‐free expression screening can serve as a viable option for rapid and precise tuning of enzyme molecules, not only for analytical purposes but also for industrial applications through large scale production using microbial cells transformed with variant genes selected from the cell‐free expression screening.

Development of a rapid and productive cell-free protein synthesis system

Due to recent advances in genome sequencing, there has been a dramatic increase in the quantity of genetic information, which has lead to an even greater demand for a faster, more parallel expression system. Therefore, interest in cell-free protein synthesis, as an alternative method for high-throughput gene expression, has been revived. In contrast toin vivo gene expression methods, cell-free protein synthesis provides a completely open system for direct access to the reaction conditions. We have developed an efficient cell-free protein synthesis system by optimizing the energy source and S30 extract. Under the optimized conditions, approximately 650 μg/mL of protein was produced after 2 h of incubation, with the developed system further modified for the efficient expression of PCR-amplified DNA. When the concentrations of DNA, magnesium, and amino acids were optimized for the production of PCR-based cell-free protein synthesis, the protein yield was comparable to that from the plasmid template.