Premium
Expression and purification of N‐terminally acetylated Ssa1 chaperone from Saccharomyces cerevisiae
Author(s) -
Griffith Alijah A.,
Holmes William
Publication year - 2019
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.2019.33.1_supplement.780.2
Subject(s) - acetylation , chaperone (clinical) , saccharomyces cerevisiae , biochemistry , proteostasis , acetyl coa , proteasome , biology , hsp90 , yeast , chemistry , microbiology and biotechnology , enzyme , heat shock protein , medicine , pathology , gene
N‐terminal acetylation leads to diversity of a given organism's proteome through constituting a cell‐wide acetylome wherein polypeptides exhibit an altered function and dynamic, acting as a basis for cell signaling pathways and allowing a given cell to react specifically to environmental stressors or cellular perturbations. The reaction of N‐terminal acetylation is catalyzed by a collection of N‐terminal acetyltransferases (NATs), wherein NAT complexes transfer acetyl groups from acetyl‐coenzyme A to the a‐amino group at amino‐terminal residues, effectively neutralizing the positive charge of the amino residue. One potential target of N‐terminal acetylation is thought to be molecular chaperone Ssa1. Endogenous to the eukaryotic Saccharomyces cerevisiae , Ssa1 is HSP70 class molecular chaperone that works in tandem with its co‐chaperone Sis1 to maintain cellular proteostasis through restoring native conformations of misfolded proteins through a transient mechanism dictated by ATP hydrolysis. Overall, the consequences of N‐terminal acetylation on the Ssa1 function and ability to maintain cellular proteostasis remains to be fully characterized. To further probe the functional and mechanistic changes associated with the N‐terminal acetylation of Ssa1, we developed an efficient, user‐friendly, one‐step purification of Ssa1 utilizing a previously constructed Protein‐A transformant strain. Our results indicate that our method is able to natively isolate high yields of pure Ssa1 from small amounts of starting material in a short period of time while allowing for the native elution of Ssa1 without compromising enzymatic activity, allowing the eluted protein to be utilized in downstream analyses to better understand interactions between Ssa1 and its co‐chaperones as well as the effects of post‐translational modifications on chaperone function. Support or Funding Information Research supported by RI‐INBRE (#P20GM103430) from NIGMS, NIH. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .