title: Investigations on the activation of recombinant microbial pro-transglutaminase: in contrast to proteinase K, dispase removes the histidine-tag.
abstract: In order to produce recombinant microbial transglutaminase (rMTG) which is free of the activating protease, dispase was used to activate the pro-rMTG followed by immobilized metal affinity chromatography (IMAC). As shown by MALDI-MS, the dispase does not only cleave the pro-sequence, but unfortunately also cleaves within the C-terminal histidine-tag. Hence, the active rMTG cannot properly bind to the IMAC material. As an alternative, proteinase K was investigated. This protease was successfully applied for the activation of purified pro-rMTG either as free or immobilized enzyme and the free enzyme was also applicable directly in the crude cell extract of E. coli. Thus, it enables a simple two-step activation/purification procedure resulting in protease-free and almost pure transglutaminase preparations. The protocol has been successfully applied to both, wild-type transglutaminase of Streptomyces mobaraensis as well as to the highly active variant S2P. Proteinase K activates the pro-rMTG without unwanted degradation of the histidine-tag. It turned out to be very important to inhibit proteinase K activity, e.g., by PMSF, prior to protein separation by SDS-PAGE.
authors.affiliation: Institute of Pharmacy, Faculty of Sciences I, Martin Luther University Halle-Wittenberg, 06099, Halle (Saale), Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Sommer
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Hertel
authors.forename: Christian E H
authors.initials: CE
authors.lastname: Schmelzer
authors.forename: Markus
authors.initials: M
authors.lastname: Pietzsch
mesh_terms: D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D006639:Histidine / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D006639:Histidine; D011503:Transglutaminases; D010450:Endopeptidases; D019286:Endopeptidase K; C023139:dispase