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Vectors for co-expression of an unrestricted number of proteins

MPG-Autoren

Scheich,  Christoph
Max Planck Society;

Soumailakakis,  Dimitri
Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons50117

Büssow,  Konrad
Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society;

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Zitation

Scheich, C., Kümmel, D., Soumailakakis, D., Heinemann, U., & Büssow, K. (2007). Vectors for co-expression of an unrestricted number of proteins. Nucleic Acids Research (London), 35(6), e43-e43. doi:10.1093/nar/gkm067.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-0010-8234-5
Zusammenfassung
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