Rapid Preparation of Truncated Transaminases using a PCR-based Cell-free Protein Synthesis System

PCR 기반의 무세포 단백질 발현 시스템을 이용한 절단 트랜스아미나제의 고속생산

  • Kwon, Yong-Chan (Department of Fine Chemical Engineering and Chemistry, School of Engineering, Chungnam National University) ;
  • Park, Kyung-Moon (Department of Chemical System Engineering, Hongik University) ;
  • Kim, Dong-Myung (Department of Fine Chemical Engineering and Chemistry, School of Engineering, Chungnam National University)
  • 권용찬 (충남대학교 공과대학 신소재공학부 정밀공업화학과) ;
  • 박경문 (홍익대학교 과학기술대학 화학시스템공학과) ;
  • 김동명 (충남대학교 공과대학 신소재공학부 정밀공업화학과)
  • Published : 2006.08.30

Abstract

In this work, we attempted the application of cell-free protein synthesis technology for the rapid generation of truncated enzymes. Truncated DNAs of a transaminase were PCR-amplified and directly expressed in cell-free protein synthesis reactions. Variants of the transaminase were rapidly prepared and analyzed for their enzymatic activity. Described method that combines the PCR and cell-free protein synthesis technologies will offer a versatile platform for the rapid generation of optimally modified protein species.

PCR증폭기술 및 무세포 단백질 발현 기술의 융합을 통하여, 여러 형태로 서열의 일부가 결손된 단백질들을 고속으로 발현할 수 있는 시스템을 구축하였다. Exonuclease 및 endonuclease에 대한 mRNA의 안정성 향상을 통하여, PCR 증폭을 통해 획득한 선형 DNA로부터의 안정적인 단백질 발현이 가능하였다. 동일한 플라스미드로부터 출발하여 수 시간 이내에 C-말단의 아미노산서열이 순차적으로 제거된 다양한 형태의 트랜스아미나제 Vf의 활성변화를 확인할 수 있었으며 이같은 기술은 각종 효소 단백질의 서열-활성 상호관계의 연구를 위한 유용한 기반을 제공할 것으로 기대된다.

Keywords

References

  1. Katzen, F., G. Chang, and W. Kudlicki (2005), The past, present and future of cell-free protein synthesis, Trends Biotechnol. 23. 150-156 https://doi.org/10.1016/j.tibtech.2005.01.003
  2. Spirin, A. S., V. I. Baranov, L. A. Ryabova, S. Y. Ovodov, and Y. B. Alakhov (1988), A continuous cell-free translation system capable of producing polypeptides in high yield, Science 242. 1162-1164 https://doi.org/10.1126/science.3055301
  3. Kim, D. M. and C. Y. Choi (1996), A semicontinuous prokaryotic coupled Transcription/Translation system using a dialysis membrane, Eur. J. Biochem. 239. 881-886 https://doi.org/10.1111/j.1432-1033.1996.0881u.x
  4. Kim, D. M. and J. R. Swartz (1999), Prolonging cell-free protein synthesis with a novel ATP regeneration system, Biotechnol. Bioeng. 66. 180-188 https://doi.org/10.1002/(SICI)1097-0290(1999)66:3<180::AID-BIT6>3.0.CO;2-S
  5. Kim, T. W., C. Y. Choi, and D. M. Kim (2006), Rapid production of milligram quantities of proteins in a batch cell-free protein synthesis system, J. Biotechnol. (in press)
  6. Ahn, J. H., H. S. Chu, T. W. Kim, I. S. Oh, C. Y. Choi, G. H. Hahn, C. G. Park, and D. M. Kim (2005), Cell-free synthesis of recombinant proteins from PCR-amplified genes at a comparable productivity to that of plasmid-based reactions, Biochem. Biophys. Res. Commun. 338. 1346-1352 https://doi.org/10.1016/j.bbrc.2005.10.094
  7. Lesley, S. A., M. A. Brow, and R. R. Burgess (1991), Use of in vitro protein synthesis from polymerase chain reaction-generated templates to study interaction of Escherichia coli transcription factors with core RNA polymerase and for epitope mapping of monoclonal antibodies, J. Biol. Chem. 266. 2632-2638
  8. Pratt, J. M., G. J. Boulnois, V. Darby, E. Orr, E. Wahle, and I. B. Holland (1981), Identification of gene products programmed by restriction endonuclease DNA fragments using an E. coli in vitro system, Nucleic Acids Res. 9. 4459-4474 https://doi.org/10.1093/nar/9.18.4459
  9. Kim, D. M., T. Kigawa, C. Y. Choi, and S. Yokoyama (1996), A highly efficient cell-free protein synthesis system fromEscherichia coli, Eur. J. Biochem. 239. 881-886 https://doi.org/10.1111/j.1432-1033.1996.0881u.x
  10. Schagger, H. and G. Jagow (1987), Tricine. sodium. dodecyl sulfate. polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa, Anal. Biochem. 166. 368-379 https://doi.org/10.1016/0003-2697(87)90587-2