Protein Science
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Protein Science (2004), 13:652-658. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplemental Research Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Goda, N.
Right arrow Articles by Shirakawa, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Goda, N.
Right arrow Articles by Shirakawa, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

The PRESAT-vector: Asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics

Natsuko Goda1, Takeshi Tenno2, Hirotoshi Takasu1, Hidekazu Hiroaki1 and Masahiro Shirakawa1

1 Graduate School of Integrated Science, Yokohama City University, Yokohama, Kanagawa 230-0045, Japan
2 Graduate School of Science and Engineering, Ehime University, Matsuyama, Ehime 790–8577, Japan

(RECEIVED September 23, 2003; FINAL REVISION November 11, 2003; ACCEPTED November 23, 2003)



Abstract

A rapid unidirectional method for cloning PCR-amplified cDNA fragments into virtually any fusion protein expression vector is described. The method, termed PRESAT-vector cloning, is based on a T-vector technique that does not require restriction endonuclease digestion of the PCR product. Subsequently, we applied a novel ORF selection method of the ligated plasmid products. This second step involves restriction endonuclease treatment that eliminates the plasmids containing an ORF in the wrong orientation prior to transformation into the bacterial host for further protein expression studies. To achieve this selection, we customized the 5'-sequence of the "rear" PCR primer corresponding to the C terminus of the protein to be expressed. The colonies harbored only the ligated products of the desired orientation at >90% efficiency. This method is applied to a GST fusion expression system, and an HTS system for soluble proteins from an expression library was tested.

Keywords: structural proteomics; recombinant fusion protein; T-vector cloning; asymmetric T-vector; unidirectional PCR cloning; high-throughput screening; glutathione-S-transferase

Abbreviations: NMR, nuclear magnetic resonance • GSH, glutathione • GST, glutathione-S-transferase • HTS, high-throughput screening • IPTG, isopropyl-D-thiogalactopyranoside • LB, Luria Broth media • HSQC, heteronuclear single quantum coherence • OD, optical density • SDS-PAGE; sodium dodecylsulfate-polyacrylamide gel electrophoresis • CDNB, 1-chloro-2,4-dinitrobenzene • PCR, polymerase chain reaction • ORF, open reading frame

Reprint requests to: Hidekazu Hiroaki, Division of Molecular Biophysics, Graduate School of Integrated Science, Yokohama City University, 1-7-29 Suehirocho, Tsurumi, Yokohama, Kanagawa 230-0045, Japan; e-mail: hiroakih{at}tsurumi.yokohama-cu.ac.jp; fax: 81-45-508-7361.

Supplemental material: See www.proteinscience.org

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03439004.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
M. Mishima, S. Wakabayashi, and C. Kojima
Solution Structure of the Cytoplasmic Region of Na+/H+ Exchanger 1 Complexed with Essential Cofactor Calcineurin B Homologous Protein 1
J. Biol. Chem., January 26, 2007; 282(4): 2741 - 2751.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Shiozawa, N. Maita, K. Tomii, A. Seto, N. Goda, Y. Akiyama, T. Shimizu, M. Shirakawa, and H. Hiroaki
Structure of the N-terminal Domain of PEX1 AAA-ATPase: CHARACTERIZATION OF A PUTATIVE ADAPTOR-BINDING DOMAIN
J. Biol. Chem., November 26, 2004; 279(48): 50060 - 50068.
[Abstract] [Full Text] [PDF]

Home page
 Protein Eng Des SelHome page
T. Tenno, N. Goda, Y. Tateishi, H. Tochio, M. Mishima, H. Hayashi, M. Shirakawa, and H. Hiroaki
High-throughput construction method for expression vector of peptides for NMR study suited for isotopic labeling
Protein Eng. Des. Sel., April 1, 2004; 17(4): 305 - 314.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 2004 by The Protein Society.