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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Publications at this Location » Publication #312416

Research Project: Immunodiagnostics to Detect Prions and Other Important Animal Pathogens

Location: Produce Safety and Microbiology Research

Title: Prion extraction methods: comparison of bead beating, ultrasonic disruption and repeated freeze-thaw methodologies for the recovery of functional renilla-prion fusion protein from bacteria

Author
item Hnasko, Robert
item Lin, Alice
item Stanker, Larry
item BALA, KUMAR - Mp Biomedical Llc
item McGarvey, Jeffery - Jeff

Submitted to: Book Chapter
Publication Type: Book / Chapter
Publication Acceptance Date: 11/17/2014
Publication Date: 1/1/2016
Citation: Hnasko, R.M., Lin, A.V., Stanker, L.H., Bala, K., McGarvey, J.A. 2016. Prion extraction methods: comparison of bead beating, ultrasonic disruption and repeated freeze-thaw methodologies for the recovery of functional renilla-prion fusion protein from bacteria. In: Micic, M., editor. Sample Preparation Techniques for Soil, Plant, and Animal Samples. New York, NY: Humana Press. p. 389-399.

Interpretive Summary: This manuscript: 1) identifies sample preparation methods to maximize recombinant protein recovery from bacteria, 2) establishes a novel renilla bioluminescent prion immunoassay and 3) outlines a strategy for the identification of anti-prion monoclonal antibodies for improved prion diagnostic immunoassays.

Technical Abstract: Molecular DNA technology allows for production of mammalian proteins in bacteria at sufficient quantities for downstream use and analysis. Variation in design and engineering of DNA expression vectors imparts selective alterations resulting in the generation of fusion proteins with intrinsic reporters or tags used to facilitate their isolation or detection. A multitude of methods are available for the disruption of bacteria that aid in the recovery of recombinant protein. Here we compare three standard methods for the disruption of bacteria (bead beating, repeated freeze-thaw and ultrasonic disruption) and evaluate the yield of a functional recombinant renilla-prion fusion protein from crude bacterial extracts. This report provides methods and guidance to maximize the yield of recombinant fusion proteins from bacteria for downstream applications