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Enhanced In Vitro Protein Synthesis Through Optimal Design of PCR Primers  

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)
Publication Information
Journal of Microbiology and Biotechnology / v.16, no.3, 2006 , pp. 355-359 More about this Journal
Abstract
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.
Keywords
In vitro protein synthesis; in vitro translation; coupled transcription/translation; linked transcription/translation; polymerase chain reaction; chloramphenicol acetyltransferase; mRNA stability;
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