Characterizing the structure and function of a cold-adapted, pepsin-like protease

Abstract

Psychrophilic enzymes can have improved functionalities in low temperatures, which may be useful for improving the efficiency or sustainability of certain industrial processes. Little is known about the structure-function relationships of cold-adapted enzymes. In particular, the existence of pepsin-like proteases which employ prosegment-catalyzed folding and autoactivation pathways in cold environments is very understudied. Comprehensive studies on folding adaptations at lower temperatures (0°C to 30°C) can provide insight into how pepsin-like proteases have adapted to natively fold and sustain life at physiologically relevant timescales. The objectives of this project were (1) to develop a high-throughput expression and purification system for a novel, cold-adapted, pepsin-like protease (RfP) originating from the Antarctic yeast Rhodotorula frigidialcoholis, (2) to identify the existence of the RfP prosegment-catalyzed autoactivation pathway, and (3) to characterize the sequence, structure, and activity profile of RfP. A truncated variant of RfP was overexpressed with a tag protein system in Escherichia coli, then purified through affinity chromatography under denaturing conditions. While expression was successful, purification issues were encountered due to denaturant-resistant aggregation of the fusion protein, resulting in reduced fraction purity and protein yield. The fusion protein was refolded then dialyzed into non-denaturing alkaline buffer (pH 9.0) to restore its native structure, then acidified (pH 5.0) to induce autoactivation. Refolding was successful when performed over extended periods of time (>24 hours), in dilute conditions, and at cold temperatures (4°C). Restoration of the native 3D structure was confirmed through visualization of the autoactivation process under acidic conditions and subsequent formation of the mature, active form of RfP (mRfP). A reverse affinity chromatography step allowed for its isolation. Assays were performed to determine the activity profiles of mRfP, and comparative analyses with pepsin were conducted. Like pepsin, mRfP was active under acidic conditions, however it was able to retain activity at lower temperatures and at higher pH conditions where pepsin was rendered inactive. This project expands the fundamental understanding of psychrophilic enzymes, from their structural adaptations to their functionality, which expands the possibility of their future use as novel biocatalysts and engineering enzymes to meet specific needs.