Controlled intracellular processing of fusion proteins by TEV protease.
Here we describe a method for controlled intracellular processing (CIP) of fusion proteins by tobacco etch virus (TEV) protease. A fusion protein containing a TEV protease recognition site is expressed in Escherichia coli cells that also contain a TEV protease expression vector. The fusion protein vector is an IPTG-inducible ColE1-type plasmid, such as a T7 or tac promoter vector. In contrast, the TEV protease is produced by a compatible p15A-type vector that is induced by tetracyclines. Not only is the TEV protease regulated independently of the fusion protein, but its expression is highly repressed in the absence of inducer. Certain fusion partners have been shown to enhance the yield and solubility of their passenger proteins. When CIP is used as a purification step, it is possible to take advantage of these characteristics while both eliminating the need for large amounts of pure protease at a later stage and possibly simplifying the purification process. Additionally, we have observed that in some cases the timing of intracellular proteolysis can affect the solubility of the cleaved passenger protein, allowing it to be directed to either the soluble or the insoluble fraction of the crude cell lysate. This method also makes it possible to quickly gauge the efficiency of proteolysis in vivo, before protein purification has begun and in vitro processing is attempted.
Kapust
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Program in Structural Biology, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland, 21702-1201, USA.
Waugh
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D020134:Catalytic Domain; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism / Q000648:ultrastructure; D005822:Genetic Vectors; D017800:Potyvirus / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012995:Solubility
D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; C099488:TEV protease