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Purification and activation of a recombinant histidine-tagged pro-transglutaminase after soluble expression in Escherichia coli and partial characterization of the active enzyme

Paper ID Volume ID Publish Year Pages File Format Full-Text
17781 42695 2008 8 PDF Available
Title
Purification and activation of a recombinant histidine-tagged pro-transglutaminase after soluble expression in Escherichia coli and partial characterization of the active enzyme
Abstract

Pro-transglutaminase from Streptomyces mobaraensis was expressed in Escherichia coli as a fusion protein carrying a C-terminal histidine tag (pro-MTG-His6). The recombinant organism was cultivated in 15 L bioreactor scale and pro-MTG-His6 was purified by immobilized metal affinity chromatography. Activation of the inactive pro-enzyme using trypsin resulted in an unexpected degradation of the transglutaminase and a concomitant loss of activity. Therefore, a set of commercially available proteases was investigated for their activation potential without destroying the target enzyme. Besides trypsin, chymotrypsin and proteinase K were found to activate but hydrolyze the (pro-MTG-His6). Cathepsin B, dispase I, and thrombin were shown to specifically hydrolyze pro-MTG-His6 without deactivation. TAMEP, the endogeneous protease from S. mobaraensis was purified for comparison and also found to activate the recombinant histidine-tagged transglutaminase without degradation. The TAMEP activated MTG-His6 was purified and characterized. The specific activity (23 U/mg) of the recombinant histidine-tagged transglutaminase, the temperature optimum (50 °C), and the temperature stability (t1/2 at 60 °C = 1.7 min) were comparable to the wild-type enzyme. A C-terminal peptide tag did neither affect the activity nor the stability but facilitated the purification. The purification of the histidine-tagged protein is possible before or after activation.

Keywords
BSA, bovine serum albumin; CV, column volume; IMAC, immobilized metal affinity chromatography; pro-MTG-His6, recombinant histidine-tagged microbial transglutaminase expressed in Escherichia coli; TG, transglutaminase; MTG, microbial transglutaminaseRecomb
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Purification and activation of a recombinant histidine-tagged pro-transglutaminase after soluble expression in Escherichia coli and partial characterization of the active enzyme
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 42, Issue 7, 5 June 2008, Pages 568–575
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us