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.
Publication types
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Evaluation Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenosine Triphosphatases / genetics
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Adenosine Triphosphatases / metabolism
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism
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Cloning, Molecular / methods*
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Escherichia coli / genetics
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Gene Expression
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Genetic Vectors / chemistry*
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Humans
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Membrane Proteins / genetics
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Membrane Proteins / isolation & purification
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Membrane Proteins / metabolism
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Protein Subunits / genetics
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Protein Subunits / isolation & purification
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Protein Subunits / metabolism
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Receptors, Autocrine Motility Factor
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Receptors, Cytokine / genetics
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Receptors, Cytokine / metabolism
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Recombinant Fusion Proteins / biosynthesis
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Recombinant Fusion Proteins / genetics
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Recombinant Proteins / biosynthesis*
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Recombinant Proteins / genetics
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Ubiquitin-Protein Ligases / genetics
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Ubiquitin-Protein Ligases / metabolism
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Valosin Containing Protein
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Vesicular Transport Proteins / genetics
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Vesicular Transport Proteins / isolation & purification
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Vesicular Transport Proteins / metabolism
Substances
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Cell Cycle Proteins
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Membrane Proteins
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Protein Subunits
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Receptors, Cytokine
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Recombinant Fusion Proteins
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Recombinant Proteins
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Vesicular Transport Proteins
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transport protein particle, TRAPP
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AMFR protein, human
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Receptors, Autocrine Motility Factor
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Ubiquitin-Protein Ligases
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Adenosine Triphosphatases
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VCP protein, human
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Valosin Containing Protein