Jin-Ho Ahn

High-throughput, combinatorial engineering of initial codons for tunable expression of recombinant proteins.

We describe a high-throughput strategy for tuning the expression of recombinant proteins through engineering their early nucleotide sequences. After randomizing the +2 and +3 codons of the target genes, each of the variant genes was isolated in vivo and subsequently expressed using in vitro protein synthesis techniques. When several hundreds of clones were examined in parallel, it was found that expression levels of target genes varied as much as 70-fold depending on the identity of the codons in the randomized region. This broad and continuous distribution of expression levels enabled the selection of specific codon arrangements for the expression of target genes at a desired level. Furthermore, codon-dependent variations in protein expression were reproduced when the same genes were expressed in vivo. Thus, we expect that the methodology reported here could be utilized as a versatile platform for rapid expression of protein molecules at modulated levels either in vitro or in vivo.

Tuning the expression level of recombinant proteins by modulating mRNA stability in a cell‐free protein synthesis system

In this work, it was discovered that the stability of mRNA in a cell-free extract could be controlled by using engineered T7 terminator sequences. Specifically, it was found that mRNA stability gradually decreased as the length of the stem structure of the T7 terminator was reduced sequentially. As a result of the controlled abundance of mRNA species, it was possible to manipulate the relative expression level of target proteins by employing the T7 terminator of adjusted stem lengths.

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.

Preparation method forEscherichia coli S30 extracts completely dependent upon tRNA addition to catalyze cell-free protein synthesis

A simple method for depletingE. coli S30 extracts of endogenous tRNA has been developed. An ethanolamine-Sepharose® column equilibrated with water selectively captured the tRNA molecules inE. coli S30 extracts. As a result, S30 extracts filtered through this column became completely dependent upon the addition of exogenous tRNA to mediate cell-free protein synthesis reactions. We anticipate that the procedures developed and described will be particularly useful forin vitro suppression reaction studies designed to introduce unnatural amino acids into protein molecules.

Use ofl-buthionine sulfoximine for the efficient expression of disulfide-containing proteins in cell-free extracts ofEscherichia coli

We have developed a technique to improve the formation of correct disulfide bonds within cell-free synthesized proteins. Via the use of a metabolic inhibitor of glutamate-cysteine ligase, the accumulation of glutathione was effectively prevented in cell-free extracts, thereby enabling the stable maintenance of redox potential for extended reaction periods. As a result, in a reaction in which a model protein contatining 9 disulfide bonds was synthesized under cell-free conditions, the final amount of active protein products was increased by 50%. The method presented in this study will provide a rapid and robust route to the high-throughput expression and screening of proteins which require multiple disulfide bonds for their activity.