[논문]Enhanced In Vitro Protein Synthesis Through Optimal Design of PCR Primers

Ahn Jin-Ho; Son Jeong-Mi; Hwang Mi-Yeon; Kim Tae-Wan; Park Chang-Kil; Choi Cha-Yong; Kim Dong-Myung

Enhanced In Vitro Protein Synthesis Through Optimal Design of PCR Primers 원문보기

Journal of microbiology and biotechnology, v.16 no.3, 2006년, pp.355 - 359

Ahn Jin-Ho (Interdisciplinary Program for Biochemical Engineering and Biotechnology, College of Engineering, Seoul National University) , Son Jeong-Mi (School of Chemical and Biological Engineering, College of Engineering, Seoul National University) , Hwang Mi-Yeon (School of Chemical and Biological Engineering, College of Engineering, Seoul National University) , Kim Tae-Wan (School of Chemical and Biological Engineering, College of Engineering, Seoul National University) , Park Chang-Kil (Department of Fine Chemical Engineering and Chemistry, Chungnam National University) , Choi Cha-Yong (Interdisciplinary Program for Biochemical Engineering and Biotechnology, College of Engineering, Seoul National University, School of Chemical and Biological Engineering, College of Engineering, Seoul National University) , Kim Dong-Myung (Department of Fine Chemical Engineering and Chemistry, Chungnam National University)

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The functional stability of mRNA is one of the crucial factors affecting the efficiency of in vitro translation. As the rapid degradation of mRNA in the cell extract (S30 extract) causes early termination of the translational reactions, extending the mRNA half-life will improve the productivity of the in vitro protein synthesis. Thus, a simple PCR-based method is introduced to increase the stability of mRNA in an S30 extract. The target genes are PCR-amplified with primers designed to make the ends of the transcribed mRNA molecule anneal to each other. When compared with normal mRNA, the mRNA with the annealing sequences resulted in an approximately 2-fold increase of protein synthesis in an in vitro translation reaction. In addition, sequential transcription and translation reactions in a single tube enabled direct protein expression from the PCR-amplified genes without any separate purification of the mRNA.

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The amount of each PCR product was determined using a spectrophotometer and agarose gel electrophoresis [5, 9]. All the mRNAs used in this study were prepared using a commercial in vitro transcription kit from Promega (Madison, WI, U.S.A.). The secondary structure of the transcribed mRNA was predicted using RNAdraw (vl.

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The enzymatic activity of the synthesized chloramphenic이 acetyltransferase (CAT) was determined by the spectrophotometric procedures described by Shaw [15]. After dil니ting a sample 1,000-fold with water, 2 μ 1 of the diluted sample was added to a 96-well plate containing 178 μ 1 of a prewarmed assay mixture (100 mM Tris-CI, pH 7.

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Enhanced In Vitro Protein Synthesis Through Optimal Design of PCR Primers 원문보기

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