Efficient and rapid affinity purification of proteins using recombinant fusion proteases.
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 which 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.
Walker
P A
PA
Protein Engineering Laboratory, National University of Singapore.
Leong
L E
LE
Ng
P W
PW
Tan
S H
SH
Waller
S
S
Murphy
D
D
Porter
A G
AG
D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular / Q000379:methods; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000378:metabolism*; D010450:Endopeptidases / Q000096:biosynthesis / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis; D010849:Picornaviridae / Q000201:enzymology*; D016133:Polymerase Chain Reaction / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification*; D013379:Substrate Specificity; D014764:Viral Proteins
D004800:Enzymes, Immobilized; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; D005982:Glutathione Transferase; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases