Abstract
Although Escherichia coli is in wide use for preparative protein expression, problems with the folding of the recombinant gene product and protein aggregation are frequently encountered. Apart from cytoplasmic expression, this is also true for secretion into the bacterial periplasm, the method of choice for the production of proteins that carry structural disulfide bonds. Here we report the construction of the helper plasmid pTUM4, which effects overexpression of four established periplasmic chaperones and folding catalysts: the thiol-disulfide oxidoreductases DsbA and DsbC that catalyze the formation and isomerization of disulfide bridges and the peptidyl-prolyl cis/trans-isomerases with chaperone activity, FkpA and SurA. pTUM4 carries a p15a origin of replication and a chloramphenicol resistance gene and, thus, it is compatible with many conventional expression vectors that use the ColEI origin and an ampicillin resistance. Its positive effects on the yield of soluble recombinant protein and the homogeneity of disulfide pattern are illustrated here using the human plasma retinol-binding protein as well as the extracellular carbohydrate recognition domain of the dendritic cell membrane receptor DC-SIGN. Hence, pTUM4 represents a novel helper vector which complements existing cytosolic chaperone coexpression plasmids and should be useful for the functional secretion of various recombinant proteins with hampered folding efficiency.
MeSH terms
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Carrier Proteins / genetics
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Carrier Proteins / metabolism
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Cell Adhesion Molecules / biosynthesis
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Cell Adhesion Molecules / chemistry
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Cell Adhesion Molecules / isolation & purification
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Escherichia coli / enzymology
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Escherichia coli / genetics*
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Escherichia coli / metabolism
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Escherichia coli Proteins / genetics
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Escherichia coli Proteins / metabolism*
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Genetic Vectors
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Humans
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Immunophilins / genetics
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Immunophilins / metabolism
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Lectins, C-Type / biosynthesis
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Lectins, C-Type / chemistry
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Lectins, C-Type / isolation & purification
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Membrane Proteins / genetics
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Membrane Proteins / metabolism
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Molecular Chaperones / genetics
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Molecular Chaperones / metabolism
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Peptidylprolyl Isomerase / genetics
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Peptidylprolyl Isomerase / metabolism
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Periplasm / metabolism*
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Plasmids / genetics
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Protein Disulfide-Isomerases / genetics
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Protein Disulfide-Isomerases / metabolism
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Protein Folding*
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Receptors, Cell Surface / biosynthesis
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Receptors, Cell Surface / chemistry
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Receptors, Cell Surface / isolation & purification
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Recombinant Proteins / biosynthesis*
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Recombinant Proteins / chemistry*
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Recombinant Proteins / isolation & purification
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Retinol-Binding Proteins / biosynthesis
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Retinol-Binding Proteins / chemistry
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Retinol-Binding Proteins / isolation & purification
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Retinol-Binding Proteins, Plasma
Substances
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Carrier Proteins
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Cell Adhesion Molecules
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DC-specific ICAM-3 grabbing nonintegrin
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Escherichia coli Proteins
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Lectins, C-Type
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Membrane Proteins
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Molecular Chaperones
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Receptors, Cell Surface
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Recombinant Proteins
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Retinol-Binding Proteins
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Retinol-Binding Proteins, Plasma
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SurA protein, E coli
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Immunophilins
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Peptidylprolyl Isomerase
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FkpA protein, E coli
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Protein Disulfide-Isomerases