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PEDS Advance Access published online on May 27, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh044
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Received December 17, 2003
Revised March 31, 2004
Accepted May 6, 2004

Article

High-throughput construction method of expression vector of peptides for NMR study suited for isotopic labeling

Takeshi Tenno 1, Natsuko Goda 2, Yukihiro Tateishi 2, Hidehito Tochio 2, Masaki Mishima 3, Hidenori Hayashi 4, Masahiro Shirakawa 2, Hidekazu Hiroaki 2*

1 Graduate School of Integrated Science, Yokohama City University, 1-7-29 Suehirocho, Tsurumi, Yokohama, Kanagawa, 230-0045 Japan; Graduate School of Science and Engineering, Ehime University, 3 Bunkyocho, Matsuyama, Ehime, 790-8577 Japan
2 Graduate School of Integrated Science, Yokohama City University, 1-7-29 Suehirocho, Tsurumi, Yokohama, Kanagawa, 230-0045 Japan
3 Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0101 Japan
4 Division of Plant Molecular Biotechnology, Cell-Free Science and Technology Research Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577 Japan

* To whom correspondence should be addressed. E-mail: hiroakih{at}tsurumi.yokohama-cu.ac.jp.


  Abstract

Fusion protein constructs for labeled peptides were generated with the 114 amino acid thioredoxin (TRX), coupled with the incorporation of a histidine tag for affinity purification. Two tandem AhdI sites were designed in the multiple cloning site of the fusion vector according to our novel unidirectional TA-cloning methodology named PRESAT-vector, allowing one-step background-free cloning of DNA fragments. Constructs were designed to incorporate the four residue sequence Ile-Asp-Gly-Arg to generate pure peptides following Factor Xa cleavage of the fusion protein. The system is efficient and cost effective for isotopic labeling of peptides for heteronuclear NMR studies. Seven peptides of varying length, including pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal peptide (VIP) and ubiquitin interacting motif (UIM), were expressed using this TRX fusion system to give soluble fusion protein constructs in all cases. Three alternative methods for preparation of DNA fragments were applied depending on the length of the peptides, such as PCR, chemical synthesis, or "semi-synthetic method", which is a combination of chemical synthesis and enzymatic extension. The ability to easily construct, express and purify recombinant peptides in a high-throughput manner will be of enormous benefit in area of biomedical research and drug discovery.

Keywords: E. coli expression system, TA-cloning, thioredoxin fusion protein, isotopic labeling, NMR


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