A genetic system yields self-cleaving inteins for bioseparations.
A self-cleaving element for use in bioseparations has been derived from a naturally occurring, 43 kDa protein splicing element (intein) through a combination of protein engineering and random mutagenesis. A mini-intein (18 kDa) previously engineered for reduced size had compromised activity and was therefore subjected to random mutagenesis and genetic selection. In one selection a mini-intein was isolated with restored splicing activity, while in another, a mutant was isolated with enhanced, pH-sensitive C-terminal cleavage activity. The enhanced-cleavage mutant has utility in affinity fusion-based protein purification. These mutants also provide new insights into the structural and functional roles of some conserved residues in protein splicing.
Wood
D W
DW
Wadsworth Center, New York State Department of Health, and School of Public Health, State University of New York at Albany, Albany, NY 12201-2002, USA.
Wu
W
W
Belfort
G
G
Derbyshire
V
V
Belfort
M
M
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000302:isolation & purification; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D007930:Leucine / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009161:Mycobacterium / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011498:Protein Precursors / Q000378:metabolism*; D019154:Protein Splicing / Q000235:genetics*; D011938:Rec A Recombinases / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification*; D013940:Thymidylate Synthase / Q000235:genetics / Q000302:isolation & purification; D014633:Valine / Q000235:genetics
D011498:Protein Precursors; D011994:Recombinant Proteins; D013940:Thymidylate Synthase; D011938:Rec A Recombinases; D010450:Endopeptidases; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II; D007930:Leucine; D014633:Valine