Engineered Active Zymogen of Microbial Transglutaminase
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Abstract
Microbial transglutaminase (MTG) has shown to be a powerful biocatalytic glue for site-specific crosslinking of a range of biomolecules and synthetic molecules, those handled with an MTG-reactive moiety. The preparation of active recombinant MTG requires the posttranslational proteolytic digestion of propeptide working as an intramolecular chaperon to assist the correct folding of MTG zymogen (MTGz) in the biosynthesis. Herein, we propose an e ngineered active z ymogen of MTG (EzMTG) that is expressed as soluble form in the host E. coli cytosol and exhibits the cross-linking activity without limited proteolysis. Based on the 3D structure of MTGz and serendipitous findings, saturated mutagenesis of K10 or Y12 in propeptide domain leads to generate several active MTGz mutants. In particular, K10D/Y12G mutant exhibited the catalytic activity comparable with a mature form. However, the expression level was low possibly due to the reduction of chaperone activity and/or the promiscuous substrate specificity of MTG, which is potentially harmful to the host cells. By contrast, soluble K10R/Y12A mutant is expressed in the cytosol of host E. coli and exhibited unique substrate-dependent reactivity toward peptidyl substrates. The quantitative analysis of the binding affinity of mutated propeptide to the active site suggested the trade-off relationship of EzMTGs between the binding affinity and the catalytic activity. Our proof-of-concept study provides insights into the design of a new biocatalyst by using the zymogen as a scaffold and will convey a potential route to the high-throughput screening of MTG mutants for bioconjugation applications.
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Thank you for your hard work on this publication! Your work has contributed to our understanding of propeptides on enzyme activity in general, and specifically on the activity of the important class of transglutaminase enzymes. Your strategy, keeping the propeptide sequence to help with protein folding and solubility while modifying propeptide residues to improve catalytic activity, could be applied across many similar cases. I hope the broader scientific field adopts and further tests this approach. I was wondering if I could ask a few questions about your expression/induction strategy. Have you considered using a different E. coli strain such as the Origami 2 (DE3) or Rosetta-gami 2 (DE3)? Were all of your different variants induced overnight at 17-18C? In some of the cases, that information is missing in your text. I also noticed the …
Thank you for your hard work on this publication! Your work has contributed to our understanding of propeptides on enzyme activity in general, and specifically on the activity of the important class of transglutaminase enzymes. Your strategy, keeping the propeptide sequence to help with protein folding and solubility while modifying propeptide residues to improve catalytic activity, could be applied across many similar cases. I hope the broader scientific field adopts and further tests this approach. I was wondering if I could ask a few questions about your expression/induction strategy. Have you considered using a different E. coli strain such as the Origami 2 (DE3) or Rosetta-gami 2 (DE3)? Were all of your different variants induced overnight at 17-18C? In some of the cases, that information is missing in your text. I also noticed the expression of some of your variants was induced by growing them in an Auto Induction Medium (without adding IPTG). What was your rationale to go that route? Thank you once more for your contribution to our understanding of propeptides and their impact on enzymatic activity!
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