Hyun Kim

Structure-based rational design of a Toll-like receptor 4 (TLR4) decoy receptor with high binding affinity for a target protein.

Repeat proteins are increasingly attracting much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural features. Nonetheless, engineering interaction interface and understanding molecular basis for affinity maturation of repeat proteins still remain a challenge. Here, we present a structure-based rational design of a repeat protein with high binding affinity for a target protein. As a model repeat protein, a Toll-like receptor4 (TLR4) decoy receptor composed of leucine-rich repeat (LRR) modules was used, and its interaction interface was rationally engineered to increase the binding affinity for myeloid differentiation protein 2 (MD2). Based on the complex crystal structure of the decoy receptor with MD2, we first designed single amino acid substitutions in the decoy receptor, and obtained three variants showing a binding affinity (KD) one-order of magnitude higher than the wild-type decoy receptor. The interacting modes and contributions of individual residues were elucidated by analyzing the crystal structures of the single variants. To further increase the binding affinity, single positive mutations were combined, and two double mutants were shown to have about 3000- and 565-fold higher binding affinities than the wild-type decoy receptor. Molecular dynamics simulations and energetic analysis indicate that an additive effect by two mutations occurring at nearby modules was the major contributor to the remarkable increase in the binding affinities.

Statistical optimization of medium composition for growth ofLeuconostoc citreum

Leuconostoc citreum is one of the representative strains ofLeuconostoc spp. that show fast growth rates in fermented vegetables. Sequential experimental designs including the Plackett-Burman design, fractional factorial design, steepest ascent analysis, central composite design and response surface methodology were introduced to optimize and improve the medium forL. citreum. Fifteen medium ingredients were examined and glucose (20 g/L), yeast extract (12.5 g/L), sodium acetate trihydrate (6.12 g/L), potassium phosphate (42.55 g/L) and dibasic ammonium citrate (4.12 g/L) were chosen as the best components to give a critical and positive effect for cell-growth. The biomass was increased to 2.79 g/L (169%), compared to the 1.65 g/L in MRS medium.

Solubilization conditions for hydrophobic membrane protein, oleosin, in soybeans

Oleosins are amphipathic proteins found in soybeans that give stability to oil-bodies by surrounding them. Furthermore, oleosins have broad biotechnological applications in protein purification and in immobilization as a tagging parther. In this study we provide the proper combinations of detergents and chaotropes for solubilization of oleosins. The solubilization effects of each detergent were compared by the results of SDS-PAGE analysis, from the density measurement of each band using an image analysis program. Among the twelve chemicals tested, 3-(decyldimethylammonio) propanesulfonale inner salt (SB 3–10), nonaethylene glycol octylphenyl ether (NP-40), and n-dodecyl-β-d-maltoside (DM) were determined to be effective for oleosin solubilization. In addition, urea, thiourea, and reducing agents played roles in enhancing the effects of the detergents. The best results were obtained with 2.5% of each detergent in combination with 4 M urea and 2 M thiourea at room temperature. The zwitterionic detergent, SB 3–10, is recommended as the best non-denaturant for the solubilization of soybean oleosins with the advantage of its easy removal by dialysis due to its low molecular weight and a high critical micelle concentration.

Comparison of endoscopic band ligation and endoclip closure of colonic perforation: technical feasibility and efficacy in an ex vivo pig model.

Recent reports have indicated several instances of successful treatment of bowel perforation by using endoscopic band ligation (EBL) when treatment with endoclipping is unsuccessful, but this salvage method has not been investigated in any prospective model. Herein we aimed to compare the technical feasibility and efficacy of EBL and endoclip use in intraluminal closure of colon perforation, in an exu2009vivo model.

Endoluminal Closure of Colon Perforation with Endoscopic Band Ligation: Technical Feasibility and Safety in an In Vivo Canine Model

Background/Aims: Endoscopic band ligation (EBL) is an accepted method in the management of variceal bleeding; however, there is little evidence on the safety and feasibility of EBL for the closure of bowel perforation. In this study, we aimed to evaluate the technical feasibility and efficacy of EBL in iatrogenic colon perforation by using a canine model. Methods: We established an iatrogenic colon perforation model by using seven beagle dogs. Longitudinal 1.5- to 1.7-cm colon perforations were created with a needle knife and an insulated-tip knife, and the perforation was subsequently closed with EBL. During a 2-week follow-up period, the animals were carefully monitored and then euthanized for pathologic examination. Results: The EBL of iatrogenic colon perforations was successful in all dogs. The mean procedure time for EBL closure with one to three bands was 191.7 seconds, and there were no immediate complications. One animal was euthanized after 3 days because of peritonitis. There were no clinical and laboratory features of sepsis or peritonitis in the remaining six animals. On necropsy, we did not find any fecal peritonitis, pericolonic abscess formation, or transmural dehiscence at the perforation site. Histopathology demonstrated inflamed granulation tissue and scar lesions replaced by fibrosis. Conclusions: EBL might be a feasible and safe method for the management of iatrogenic colon perforations in an in vivo model.