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Published online before print May 7, 2004, 10.1110/ps.04644504
Protein Science (2004), 13:1698-1703. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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FOR THE RECORD

Two-promoter vector is highly efficient for overproduction of protein complexes

Kyung-Jin Kim1, Han-Eol Kim, Kwang-Hoon Lee, Wondeok Han, Min-Ju Yi, Jinseok Jeong and Byung-Ha Oh

Center for Biomolecular Recognition and Division of Molecular and Life Science, Department of Life Sciences, Pohang University of Science and Technology, Pohang, Kyungbuk, 790-784, Korea

(RECEIVED January 19, 2004; FINAL REVISION February 19, 2004; ACCEPTED February 20, 2004)



Abstract

The use of bicistronic vectors, which contain two target genes under one promoter, has been the most common practice for the heterologous production of binary protein complexes. The major problem of this method is the much lower expression of the second gene compared with that of the first gene next to the promoter. We tested a simple idea of whether inclusion of an additional promoter in front of the second gene may remove the problem. Compared with bicistronic vectors, corresponding two-promoter vectors yielded four to nine times larger amounts of the complexes between BCL-2 family proteins, BCL-XL:BAD, BCL-XL:BIM-S, and CED-9:EGL-1 in bacterial cells as a result of significantly increased expression of the second genes in a manner independent of the order of the target genes. With the two-promoter system, we produced two other complexes in large quantity suitable for extensive crystallization trial. The method does not accompany any technical disadvantages, and represents a significant improvement from the conventional method, which should enjoy wide application for the coexpression of binary or higher order protein complexes by extension.

Keywords: protein expression system; two-promoter; protein complex; apoptosis

Reprint requests to: Byung-Ha Oh, Center for Biomolecular Recognition and Division of Molecular and Life Science, Department of Life Sciences, Pohang University of Science and Technology, Pohang, Kyungbuk, 790-784, Korea; e-mail: bhoh{at}postech.ac.kr; fax: 82-54-279-2199; or Kyung-Jin Kim, Pohang Accelerator Laboratory, Pohang, Kyungbuk, 790-784, Korea; e-mail: kkj{at}postech.ac.kr; fax: 82-54-2119-1599.

1 Present address: Pohang Accelerator Laboratory, Pohang, Kyungbuk, 790-784, Korea.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04644504.


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