Expression and rapid purification of highly active hexahistidine-tagged guinea pig liver transglutaminase.
Tissue transglutaminase has been identified as a contributor to a wide variety of diseases, including cataract formation and Celiac disease. Guinea pig tissue transglutaminase has a very broad substrate specificity and therefore is useful for kinetic studies using substrate analogues. Here, we report the expression in Escherichia coli of a hexahistidine-tagged guinea pig liver tissue transglutaminase (His(6)-tTGase) allowing rapid purification by immobilized-metal affinity chromatography. Using this procedure we have obtained the highest reported specific activity (17 U/mg) combined with a high yield (22 mg/L of culture) for recombinant TGase using a single-step purification protocol. Using two independent spectrophotometric assays, we determined that the K(m) value of the recombinant enzyme with the substrate Cbz-Gln-Gly is in the same range as values reported in the literature for the native enzyme. We have thus developed a rapid and reproducible protocol for the preparation of high quality tissue TGase.
Gillet
Steve M F G
SM
Département de Chimie, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, Québec, Canada H3C 3J7.
Chica
Roberto A
RA
Keillor
Jeffrey W
JW
Pelletier
Joelle N
JN
D000818:Animals; D002846:Chromatography, Affinity; D003470:Culture Media; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006168:Guinea Pigs / Q000235:genetics*; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D011503:Transglutaminases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D003470:Culture Media; D011994:Recombinant Proteins; D006639:Histidine; D011503:Transglutaminases