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Applied and Environmental Microbiology, February 2004, p. 712-721, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.712-721.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Modular Broad-Host-Range Expression Vectors for Single-Protein and Protein Complex Purification

Barna D. Fodor,1 Ákos T. Kovács,1,2 Róbert Csáki,2 Éva Hunyadi-Gulyás,3 Éva Klement,3 Gergely Maróti,1,2 Lívia S. Mészáros,1,2 Katalin F. Medzihradszky,3 Gábor Rákhely,1,2 and Kornél L. Kovács1,2*

Institute of Biophysics,1 Mass Spectrometry Facility, Biological Research Center, Hungarian Academy of Sciences,3 Department of Biotechnology, University of Szeged, H-6726 Szeged, Hungary2

Received 7 July 2003/ Accepted 4 November 2003

A set of modular broad-host-range expression vectors with various affinity tags (six-His-tag, FLAG-tag, Strep-tag II, T7-tag) was created. The complete nucleotide sequences of the vectors are known, and these small vectors can be mobilized by conjugation. They are useful in the purification of proteins and protein complexes from gram-negative bacterial species. The plasmids were easily customized for Thiocapsa roseopersicina, Rhodobacter capsulatus, and Methylococcus capsulatus by inserting an appropriate promoter. These examples demonstrate the versatility and flexibility of the vectors. The constructs harbor the T7 promoter for easy overproduction of the desired protein in an appropriate Escherichia coli host. The vectors were useful in purifying different proteins from T. roseopersicina. The FLAG-tag-Strep-tag II combination was utilized for isolation of the HynL-HypC2 protein complex involved in hydrogenase maturation. These tools should be useful for protein purification and for studying protein-protein interactions in a range of bacterial species.

* Corresponding author. Mailing address: Department of Biotechnology, University of Szeged, H-6726 Szeged, Temesvári krt. 62, Hungary. Phone: 36 62 544 351. Fax: 36 62 544 352. E-mail: kornel{at}nucleus.szbk.u-szeged.hu.

Applied and Environmental Microbiology, February 2004, p. 712-721, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.712-721.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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