Jong-In Won

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

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.

The effects of supplementing specific amino acids on the expression of elastin-like polypeptides (ELPs)

Elastin-like polypeptides (ELPs) made from the repeating pentapeptides (Val-Pro-Gly-Xaa-Gly) are protein based biopolymers that contain useful properties, including the ability to self-assemble, biocompatibility, and stimuli sensitivity. However, due to the repeated consumption of specific amino acids, long ELPs generally have low expression yields in in vitro and in vivo systems. This is because of the lack of specific amino acids during the translation process. In this study, ELP fusion proteins of various lengths were prepared by recursive directional ligation (RDL) and expressed in a cell-free protein synthesis system. By measuring TCA-precipitated radioactivity with a liquid scintillation counter, their expression profiles were investigated. The expression levels of an ELP fusion protein were improved by almost 2-fold by adding specific amino acids. Additionally, we determined that the amount of increase in expression levels depends on the length of the ELPs. This study suggests a useful strategy to improve the yield of longer repetitive polypeptides such as ELPs or silk-like polypeptides (SLPs).

Recent advances in DNA sequencing by End-Labeled Free-Solution Electrophoresis (ELFSE)

End-Labeled Free-Solution Electrophoresis (ELFSE) is a new technique that is a promising bioconjugate method for DNA sequencing (or separation) and genotyping by both capillary and microfluidic device electrophoresis. Because ELFSE enables high-resolution electrophoretic separation in aqueous buffer alone (i.e., without a polymer matrix), it eliminates the need to load viscous polymer networks into electrophoresis microchannels. To achieve microchannel DNA separations with high performance, ELFSE requires monodisperse perturbing entities (i.e., drag-tags), which create a large amounts of frictional drag when pulled behind DNA during free-solution electrophoresis, and which have other properties suitable for microchannel electrophoresis. In this article, the theoretical concepts of ELFSE and the required characteristics of the drag-tag molecules for the ultimate performance of ELFSE are reviewed. Additionally, the merits and limitations of current drag-tags are also discussed in the context of recent experimental data of ELFSE separation (or sequencing).

Influence of Vitreoscilla hemoglobin gene expression on 2,3-butanediol production in Klebsiella oxytoca

A hemoglobin-like protein is a protein that aids in the delivery of oxygen in microbes in a similar fashion as hemoglobins in plants and animals. In this study, expression of Vitreoscilla hemoglobin (VHb) was used to improve the growth of Klebsiella oxytoca, and consequently, the 2,3-butanediol (2,3-BDO) production. The Vitreoscilla hemoglobin gene (vgb) was cloned into the expression vector pET-24a, and the recombination plasmid was then transformed in Klebsiella oxytoca ATCC43863. The efficiency of 2,3-BDO production and cell growth was analyzed by placing VHb expression under the control of the nar and luxS promoters instead of the T7 in Klebsiella oxytoca. Fermentation experiments were performed for comparison with the wild type under conditions of high/low aeration for the optimization of VHb-expressing cells in a 500 mL shaking flask and a 5 L fermenter. Moreover, cultivations were performed with addition of acetic acid to the culture media to study the effects of acetic acid on 2,3-BDO production, as acetic acid is known to induce 2,3-BDO production. The results indicated a 10% increase of cell growth in the presence of VHb, while 2,3-BDO production was further improved by the addition of acetic acid. In particular, the fed-batch fermentation of Klebsiella oxytoca LV with acetic acid added exhibited increase of the dry cell weight from 11.9 to 14.0 g/L, and improvement of the 2,3-BDO production from 34.9 to 49.4 g/L in 40 h.

Validation of 4 type-STR analysis for identification of 50 Korean

Short tandem repeat (STR) analysis provides genetic fingerprinting of individuals and is an indispensable technique for forensic human identification. Recently, this technique has been used in social areas, such as the identification of The Korean War, descendants of national merit, and missing children. STR analysis is performed by analyzing iteration number of repeating bases in the human genome, and currently FBI provides the Combined DNA Index System (CODIS) based on DNA databases. Among them, we used the autosomal short tandem repeats of loci D13S317, D16S539, D21S11, and amelogenin to validate this technique for identification. The samples were collected from unrelated 50 Korean individuals, and 4 STR loci of these samples were analyzed by ABI 3130 genetic analyzer. We demonstrated that 47 samples out of 50 were classified completely with only 4 STR markers, and perfect sex identification could be accomplished with amelogenin analysis.

An analysis of the concentration change of intermediate metabolites by gene manipulation in fatty acid biosynthesis.

In this report, concentration of malonic acid and acetic acid produced in Escherichia coli were investigated by the expression of acetyl-CoA carboxylase genes (accs) and a malonyl-CoA:ACP transacylase gene (fabD). Both malonyl-CoA and acetyl-CoA are essential intermediate metabolites in the fatty acid biosynthetic pathway, and are reversibly transformed to malonic acid and acetic acid, respectively in the cell. Acetyl-CoA is converted to malonic-CoA by acetyl-CoA carboxylases (Accs), which are composed of 3 different subunits (AccA, AccB, and AccC), and the resulting malonyl-CoA is then converted to malonyl-[acp] by malonyl-CoA:ACP transacylase (FabD). In this study, these genes were separately cloned, and the influences of overexpression of 4 different genes on the concentration of malonic acid and acetic acid were analyzed. Compared with the wild type E. coli, a recombinant strain containing 3 acc genes together showed a 41.03% enhanced malonic acid production, and a 4.29-fold increased ratio of malonic acid to acetic acid.

Synthesis and characterization of an ELP-conjugated liposome with thermo-sensitivity for controlled release of a drug

Liposome, one of various drug carriers, has been extensively studied as an inert carrier for the delivery of protein, DNA, and biologically active agents into cells. Recently, much effort has been directed to the development of stimuli-sensitive liposomes that are able to respond to certain internal or external stimuli, such as, pH, electricity, temperature, magnet, or light. Among them, to obtain liposomes which release the contents in response to ambient temperature, liposomes have been modified with chemically synthetic polymers having various lower critical solution temperatures (LCST). In this study, instead of chemically synthetic polymers, a biologically produced elastin-like polypeptide (ELP), which was composed of oligomeric repeats of the pentapeptide sequence (Val-Pro-Gly-Val- Gly), was used for endowing the liposome with thermosensitivity. A model drug was encapsulated in the ELPconjugated liposomes and the release behavior of the drug caused by the liposome disruption due to the aggregation of ELPs was investigated. In addition, conjugation of ELP to liposome was identified with Fourier Transformed Infrared (FT-IR) and Scanning Electron Microscope (SEM) analyses.