Structural investigation of APRs to improve the solubility of outer membrane protease (PgtE) from Salmonella enterica serotype typhi- A multi-constraint approach

Abstract

Outer membrane proteins were playing a crucial role on the several functions controlled by cell membranes even though they are not naturally expressed at higher levels. In order to obtain biologically active protein, the denaturation of these inclusion bodies must be optimized using chaotropic agents. Hence, this study focuses on improving the yield of Outer Membrane Protease (PgtE) from Salmonella enterica serotype Typhi (S. Typhi) using chaotropes and additives. Denaturation methods were tried with various pH, detergents, and reducing agents were used to optimize the solubility of PgtE with biologically active form. Due to the aggregation, we failed to achieve the maximum yield of PgtE. Consequently, we predicted 9 Aggregation Prone Regions (APRs) in PgtE, which are mutated by known structural Gatekeepers. We calculated the Aggregation Index (AI) of PgtE with 10 mM of aspartic acid as an additive in optimized buffer. In addition, the mutations at specific positions within the protein structure can act as APRs suppressors without affecting protein stability with CABS flex dynamics. The multiple sequence analysis demonstrate that aspartic acid is appropriate denaturing additive for other Gram-negative pathogens of Omptin family. Keywords: Salmonella enteric serotype typhi, PgtE, APRs, Solubilization, CABS flex, Structural gatekeepers