optimized-production-analysis-recombinant-protein-filled-vesicles

Optimized Production and Analysis of Recombinant Protein-Filled Vesicles from <em>E. coli</em>

This innovative system, using a short peptide tag, that exports multiple recombinant proteins in membrane bound vesicles from E. coli, provides an ...

University of Kent

Maintaining membrane integrity is of paramount importance to the survival of bacteria as the membrane is the site of multiple crucial cellular processes including energy generation, nutrient uptake and antimicrobial efflux. The DedA family of integral membrane proteins are widespread in bacteria and are associated with maintaining the integrity of the membrane. In addition, DedA proteins have been linked to resistance to multiple classes of antimicrobials in various microorganisms. Therefore, the DedA family are attractive targets for the development of new antibiotics. Despite DedA family members playing a key physiological role in many bacteria, their structure, function and physiological role remain unclear. To help illuminate the structure of the bacterial DedA proteins, we performed substituted cysteine accessibility method (SCAM) analysis on the most comprehensively characterized bacterial DedA protein, YqjA from . By probing the accessibility of 15 cysteine residues across the length of YqjA using thiol reactive reagents, we mapped the topology of the protein. Using these data, we experimentally validated a structural model of YqjA generated using evolutionary covariance, which consists of an α-helical bundle with two re-entrant hairpin loops reminiscent of several secondary active transporters. In addition, our cysteine accessibility data suggest that YqjA forms an oligomer wherein the protomers are arranged in a parallel fashion. This experimentally verified model of YqjA lays the foundation for future work in understanding the function and mechanism of this interesting and important family.

Topological analysis of a bacterial DedA protein associated with alkaline tolerance and antimicrobial resistance.

We describe an innovative system that exports diverse recombinant proteins in membrane-bound vesicles from . These recombinant vesicles compartmentalize proteins within a micro-environment that enables production of otherwise challenging insoluble, toxic, or disulfide-bond containing proteins from bacteria. The release of vesicle-packaged proteins supports isolation from the culture and allows long-term storage of active protein. This technology results in high yields of vesicle-packaged, functional proteins for efficient downstream processing for a wide range of applications from discovery science to applied biotechnology and medicine.

Article	Year
Topological analysis of a bacterial DedA protein associated with alkaline tolerance and antimicrobial resistance. Microbiology (Reading, England)\| PubMed ID: 34914576	2021
High-yield vesicle-packaged recombinant protein production from . Cell reports methods\| PubMed ID: 36936078	2023

Bree R. Streather

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