The use of recombinant fusion proteases in the affinity purification of recombinant proteins.
In the affinity purification of recombinant fusion proteins, the rate-limiting step is usually the efficient proteolytic cleavage and removal of the affinity tail and the protease from the purified recombinant protein. We have developed a rapid, convenient, and efficient method of affinity purification that can overcome this limitation. In one example of the method, the protease 3C from a picornavirus (3Cpro), which cleaves specific sequences containing a minimum of 6-7 amino acids, has been expressed as a fusion with glutathione S-transferase. The resultant recombinant "fusion protease" cleaves fusion proteins bearing (from the amino-terminus) the same affinity tail as the fusion protease, a 3Cpro cleavage recognition site, and the recombinant protein of interest. The recombinant protein is purified in a single chromatographic step, which removes both the affinity tail and the fusion protease. The advantages over existing methods include much improved specificity of proteolytic cleavage, complete removal of the protease and the affinity tail in one step, and the option of adding any desired amount of fusion protease to ensure efficient cleavage. The potential flexibility of the method is shown by the use of various affinity tails and alternative fusion proteases.
Leong
L E
LE
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, USA.
D000086042:3C Viral Proteases; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification*; D014764:Viral Proteins
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; D005982:Glutathione Transferase; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases; C057400:picornain 2A, Picornavirus