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Molecular characterization of C. elegans Hsp70‐1 and the effects of polyhistidine tagging on purification yield and ATPase activity
Author(s) -
Odunuga Odutayo O.,
Polvadore Elton I.,
Choi Ka H.,
Bollinger Sarah A.
Publication year - 2012
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.26.1_supplement.963.11
Subject(s) - caenorhabditis elegans , hsp70 , heat shock protein , biology , biochemistry , escherichia coli , recombinant dna , atpase , mutant , enzyme , gene , microbiology and biotechnology , chemistry
Caenorhabditis elegans has been used as a model organism to study the roles of molecular chaperones in cellular processes. C. elegans heat shock protein 70‐1 gene, CeHsp70‐1 , is the first of the 13‐member Hsp70 family genes identified in the organism. The protein product of this gene, CeHsp70‐1, has been shown to play an important role in conferring thermo‐tolerance and longevity on C. elegans . Here, we present the results of the first work to over‐express, purify and characterize the ATP hydrolyzing activity of a member of the C. elegans Hsp70s. Recombinant CeHsp70‐1 was found to be highly expressed and sufficiently soluble in Escherichia coli . The protein was purified to homogeneity using a combination of nickel affinity, ion exchange and size‐exclusion chromatography. Kinetic properties of the basal ATPase activity of the enzyme in a low‐salt buffer were determined using a colorimetric assay. The specific activity, k m and k cat values obtained for CeHsp70‐1 were 25 nmol/min/mg, 50 μM and 0.28 min −1 , respectively. The k cat of the protein was found to be similar to that of heat shock cognate 70 (Hsc70) and binding immunoglobulin protein (BiP). At low concentrations, CeHsp70‐1 existed mostly in its monomeric form. We show that direct N‐terminal (His) 6 ‐tagging of CeHsp70‐1 lowers purification yield and affects ATPase activity.