Nucleic Acids Research Advance Access published online on February 20, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm067
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Molecular Biology |
Vectors for co-expression of an unrestricted number of proteins
1Max Planck Institute for Molecular Genetics, Department of Vertebrate Genomics, Ihnestr. 63-73, 14195 Berlin, Germany, 2Max Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13125 Berlin, Germany and 3Free University of Berlin, Institute of Chemistry and Biochemistry, Takustraße 6, 14195 Berlin, Germany
*To whom correspondence should be addressed. Tel: +49 30 9406 2983; Fax: +49 30 9406 2925; E-mail: buessow{at}molgen.mpg.de
Received November 10, 2006. Revised January 17, 2007. Accepted January 23, 2007.
A vector system is presented that allows generation of E. coli co-expression clones by a standardized, robust cloning procedure. The number of co-expressed proteins is not limited. Five ‘pQLink’ vectors for expression of His-tag and GST-tag fusion proteins as well as untagged proteins and for cloning by restriction enzymes or Gateway cloning were generated. The vectors allow proteins to be expressed individually; to achieve co-expression, two pQLink plasmids are combined by ligation-independent cloning. pQLink co-expression plasmids can accept an unrestricted number of genes. As an example, the co-expression of a heterotetrameric human transport protein particle (TRAPP) complex from a single plasmid, its isolation and analysis of its stoichiometry are shown. pQLink clones can be used directly for pull-down experiments if the proteins are expressed with different tags. We demonstrate pull-down experiments of human valosin-containing protein (VCP) with fragments of the autocrine motility factor receptor (AMFR). The cloning method avoids PCR or gel isolation of restriction fragments, and a single resistance marker and origin of replication are used, allowing over-expression of rare tRNAs from a second plasmid. It is expected that applications are not restricted to bacteria, but could include co-expression in other hosts such as Bacluovirus/insect cells.