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| Item Type: | Article |
|---|---|
| Title: | Vectors for co-expression of an unrestricted number of proteins |
| Creators Name: | Scheich, C. and Kuemmel, D. and Soumailakakis, D. and Heinemann, U. and Buessow, K. |
| Abstract: | 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. |
| Keywords: | Adenosine Triphosphatases, Cell Cycle Proteins, Molecular Cloning, Escherichia coli, Gene Expression, Genetic Vectors, Membrane Proteins, Protein Subunits, Cytokine Receptors, Recombinant Fusion Proteins, Recombinant Proteins, Ubiquitin-Protein Ligases, Vesicular Transport Proteins |
| Source: | Nucleic Acids Research |
| ISSN: | 0305-1048 |
| Publisher: | Oxford University Press |
| Volume: | 35 |
| Number: | 6 |
| Page Range: | e43 |
| Date: | March 2007 |
| Official Publication: | https://doi.org/10.1093/nar/gkm067 |
| PubMed: | View item in PubMed |
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