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Title: Role of Lipopolysaccharide in protecting OmpT from autoproteolysis during in vitro refolding
Authors: Sinsinbar, Gaurav
Gudlur, Sushanth
Metcalf, Kevin J.
Mrksich, Milan
Nallani, Madhavan
Liedberg, Bo
Keywords: Science::Biological sciences
Issue Date: 2020
Source: Sinsinbar, G., Gudlur, S., Metcalf, K. J., Mrksich, M., Nallani, M., & Liedberg, B. (2020). Role of Lipopolysaccharide in protecting OmpT from autoproteolysis during in vitro refolding. Biomolecules, 10(6), 922-. doi:10.3390/BIOM10060922
Project: MOE2018-T2-1-025 
NSF NNCI-1542205 
P41 GM108569 
Journal: Biomolecules 
Abstract: Outer membrane protease (OmpT) is a 33.5 kDa aspartyl protease that cleaves at dibasic sites and is thought to function as a defense mechanism for E. coli against cationic antimicrobial peptides secreted by the host immune system. Despite carrying three dibasic sites in its own sequence, there is no report of OmpT autoproteolysis in vivo. However, recombinant OmpT expressed in vitro as inclusion bodies has been reported to undergo autoproteolysis during the refolding step, thus resulting in an inactive protease. In this study, we monitor and compare levels of in vitro autoproteolysis of folded and unfolded OmpT and examine the role of lipopolysaccharide (LPS) in autoproteolysis. SDS-PAGE data indicate that it is only the unfolded OmpT that undergoes autoproteolysis while the folded OmpT remains protected and resistant to autoproteolysis. This selective susceptibility to autoproteolysis is intriguing. Previous studies suggest that LPS, a co-factor necessary for OmpT activity, may play a protective role in preventing autoproteolysis. However, data presented here confirm that LPS plays no such protective role in the case of unfolded OmpT. Furthermore, OmpT mutants designed to prevent LPS from binding to its putative LPS-binding motif still exhibited excellent protease activity, suggesting that the putative LPS-binding motif is of less importance for OmpT’s activity than previously proposed.
ISSN: 2218-273X
Schools: School of Materials Science and Engineering 
Organisations: Northwestern University 
Rights: © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (
Fulltext Permission: open
Fulltext Availability: With Fulltext
Appears in Collections:MSE Journal Articles

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