Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element.
A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), beta-mercaptoethanol (beta-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.
Chong
S
S
New England Biolabs, Beverly, MA 01915, USA.
Mersha
F B
FB
Comb
D G
DG
Scott
M E
ME
Landry
D
D
Vence
L M
LM
Perler
F B
FB
Benner
J
J
Kucera
R B
RB
Hirvonen
C A
CA
Pelletier
J J
JJ
Paulus
H
H
Xu
M Q
MQ
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry; D002686:Chitin; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D058619:Maltose-Binding Proteins; D008722:Methods; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D019154:Protein Splicing; D006180:Proton-Translocating ATPases / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification*; D013696:Temperature; D025262:Vacuolar Proton-Translocating ATPases
D000345:Affinity Labels; D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D011994:Recombinant Proteins; D002686:Chitin; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases