Vectors for co-expression of an unrestricted number of proteins.
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.
Scheich
Christoph
C
Max Planck Institute for Molecular Genetics, Department of Vertebrate Genomics, Berlin, Germany.
Kümmel
Daniel
D
Soumailakakis
Dimitri
D
Heinemann
Udo
U
Büssow
Konrad
K
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D060285:Receptors, Autocrine Motility Factor; D018121:Receptors, Cytokine / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism; D000074405:Valosin Containing Protein; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D018797:Cell Cycle Proteins; D008565:Membrane Proteins; D021122:Protein Subunits; D018121:Receptors, Cytokine; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D033921:Vesicular Transport Proteins; C406327:transport protein particle, TRAPP; C555000:AMFR protein, human; D060285:Receptors, Autocrine Motility Factor; D044767:Ubiquitin-Protein Ligases; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein