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Isolation and Purification of Functional Amyloid Proteins in Haloferax volcanii
Author(s) -
Sawyer Erica,
Kozodoy Lisa,
Raimer Heather,
Olendzenski Lorraine,
Marano Nadia
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.04450
Subject(s) - thioflavin , haloferax volcanii , chemistry , amyloid (mycology) , biochemistry , biofilm , differential centrifugation , prion protein , ultracentrifuge , bacteria , chromatography , archaea , biology , genetics , medicine , inorganic chemistry , disease , pathology , gene , alzheimer's disease
Haloferax volcanii , an extremophile species of Archaea, is a microorganism that incorporates functional amyloids into its biofilms; these proteins are proposed to contribute to the structural stability of this matrix. We developed a protocol based on the procedure described by Jordal et al. (2009) to isolate these proteins via differential centrifugation. The presence of amyloid proteins was monitored and quantified throughout this process using Thioflavin T, a molecule that can selectively quantify amyloids by fluorescing when bound to their cross‐beta motif. These results showed that a four‐week growth period was ideal and resulted in the highest amyloid fiber yield. The final supernatant contained the majority of the amyloid protein and had percent recoveries ranging from 41.2% to 63.4%, while the final pellet had recoveries from 6.3%‐18.1%. To further improve the efficiency of this procedure, we are utilizing the detergent Triton X‐100 during the isolation process. Preliminary findings show that this detergent slightly increases the amyloid percent recovery. Isolated proteins will ultimately be purified using preparatory SDS‐PAGE for atomic force microscopy imaging, deaggregated into their monomeric forms using formic acid, and sequenced to gain further information about their structure. Support or Funding Information SLU Fellowship Program & Strading Research Fund