http://chineseinput.net/에서 pinyin(병음)방식으로 중국어를 변환할 수 있습니다.
변환된 중국어를 복사하여 사용하시면 됩니다.
Christy Catherine,오수진,이경호,민승의,원종인,윤형돈,김동명 한국생물공학회 2015 Biotechnology and Bioprocess Engineering Vol.20 No.3
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.
박유진,( Christy Catherine ),( Devi Kasi ),이경호,신승민,김용성,김동명 한국공업화학회 2016 한국공업화학회 연구논문 초록집 Vol.2016 No.0
In this study we attempted to express the scFv antibodies that had been engineered to penetrate into the cells and give signal through split GFP complementation. Cell-free protein synthesis system provides a high accessibility to the translational mechanism, thereby providing optimal environments to increase the productivity and solubility of scFv. To assist the formation of correct disulfide bonds, we have changed redox conditions of the cell extract and reaction mixture using GSSG/GSH redox buffer. To increase the solubility, we used a cell extract enriched with DsbA, DsbC, FkpA and SurA. We also used the leader sequence ubiquitin,in front of the target scFv and achieved a high level expression of functional scFv antibodies. The functionality has confirmed by reading the fluorescence signal of transfected cells in a black plate assay.
이경호,( Christy Catherine ),( Devi Kasi ),이승원,신현일,김유정,주정원,김동명 한국공업화학회 2015 한국공업화학회 연구논문 초록집 Vol.2015 No.0
Screening of suitable antigen is an important step in the development of diagnostic and therapeutic tools in the medical field. This study describes high-throughput expression screening of parasite based on the techniques of cell-free protein synthesis. The libraries of Plasmodium vivax and Clonorchis sinensis genes were PCR-amplified and immobilized on streptavidin agarose beads. Through dual functionalization of the agarose beads to capture 6xhistidine-tagged proteins as well as the biotinylated PCR products, when expressed in a reaction mixture for cell-free synthesis,template DNA and the expressed protein were in situ immobilized on the same beads. This approach of in situ expression and isolation enables streamlined recovery and analysis of cell-free synthesized proteins and also allows facile identification of the genes coding antigenic proteins through direct PCR of the microbead-bound DNA.
이경호,김동명,Christy Catherine 한국공업화학회 2016 Journal of Industrial and Engineering Chemistry Vol.37 No.-
Replacement of canonical amino acids with unnatural amino acids (UAAs) can provide proteins withnovel physicochemical properties and biological functions. In this study, as an alternative option toconventional cell-based methods, we used a cell-free protein synthesis system as a flexible platform forfacile and efficient production of UAA-containing proteins. We designed a cell-free protein synthesissystem derived from the extract of Escherichia coli cells to maximize the selective incorporation of UAAsinto the protein structure. First, for the purpose of avoiding competitive incorporation of canonicalamino acids and UAAs, the cell extract was extensively washed using a diafiltration process to removeresidual amino acids, thereby making the protein synthesis reaction completely dependent upon theexogenous addition of amino acids. In addition, the relatively low affinity of UAAs for cognate aminoacyl-tRNA synthetase was kinetically overcome by increasing the concentration of UAAs to nonphysiologicallevels. As a result of these modifications of the cell-free synthesis systems, we were able to produce UAAcontainingproteins at comparable yields to those of proteins made of canonical amino acids.