Purification of proteins fused to either the amino or carboxy terminus of the Mycobacterium xenopi gyrase A intein.
The Mycobacterium xenopi gyrase A mini-intein has been engineered to yield a controllable N-terminal or C-terminal, single-splice-junction autocleavage element. When combined with an affinity tag, these modified mini-inteins can be used to purify target proteins after a single combined chromatography/cleavage step. Cleavage at the intein N terminus was induced with thiol reagents, while cleavage at the intein C terminus was induced by a temperature shift to 16 degrees-25 degrees C. Different preferences for the residue immediately preceding the intein were observed during thiol-induced, N-terminal splice-junction cleavage of the M. xenopi gyrase A mini-intein vs. the Saccharomyces cerevisiae vacuolar ATPase, subunit A (VMA) intein present in the IMPACT purification system. Furthermore, the M. xenopi gyrase A mini-intein C-terminal autocleavage vector allows isolation of polypeptides with N-terminal cysteine residues that are active in the Intein Mediated Protein Ligation method of protein semisynthesis.
Southworth
M W
MW
New England Biolabs, Beverly, MA, USA.
Amaya
K
K
Evans
T C
TC
Xu
M Q
MQ
Perler
F B
FB
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli; D037241:Mannose-Binding Lectins; D019912:Mycobacterium xenopi / Q000201:enzymology*; D015202:Protein Engineering; D019154:Protein Splicing; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013696:Temperature; D013879:Thioredoxins / Q000737:chemistry; D014335:Tropomyosin / Q000737:chemistry
D001426:Bacterial Proteins; D002352:Carrier Proteins; D037241:Mannose-Binding Lectins; D011993:Recombinant Fusion Proteins; D014335:Tropomyosin; D013879:Thioredoxins; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II