Jung-Won Keum

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.

Expression Screening of Fusion Partners from an E. coli Genome for Soluble Expression of Recombinant Proteins in a Cell-Free Protein Synthesis System

While access to soluble recombinant proteins is essential for a number of proteome studies, preparation of purified functional proteins is often limited by the protein solubility. In this study, potent solubility-enhancing fusion partners were screened from the repertoire of endogenous E. coli proteins. Based on the presumed correlation between the intracellular abundance and folding efficiency of proteins, PCR-amplified ORFs of a series of highly abundant E. coli proteins were fused with aggregation-prone heterologous proteins and then directly expressed for quantitative estimation of the expression efficiency of soluble translation products. Through two-step screening procedures involving the expression of 552 fusion constructs targeted against a series of cytokine proteins, we were able to discover a number of endogenous E. coli proteins that dramatically enhanced the soluble expression of the target proteins. This strategy of cell-free expression screening can be extended to quantitative, global analysis of genomic resources for various purposes.

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.

Combinatorial, selective and reversible control of gene expression using oligodeoxynucleotides in a cell-free protein synthesis system†

Herein we describe the methods for selective and reversible regulation of gene expression using antisense oligodeoxynucleotides (ODNs) in a cell‐free protein synthesis system programmed with multiple DNAs. Either a complete shut down or controlled level of gene expression was attained through the antisense ODN‐mediated regulation of mRNA stability in the reaction mixture. In addition to the primary control of gene expression, we also demonstrate that the inhibition of protein synthesis can be reversed by using an anti‐antisense ODN sequence that strips the antisense ODN off the target sequence of mRNA. As a result, sequential additions of the antisense and anti‐antisense ODNs enabled the stop‐and‐go expression of protein molecules. Through the on‐demand regulation of gene expression, presented results will provide a versatile platform for the analysis and understanding of the complicated networks of biological components. Biotechnol. Bioeng. 2009;102: 577–582.