Caenorhabditis elegans Hsp70-1 expresses highly in bacteria, is sufficiently soluble, and has a catalytic constant similar to Hsc70 and BiP.
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 (CeHsp70-1) is the first of the 13-member Hsp70 family genes identified so far 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 (V(max) per mg protein), K(m) and k(cat) values obtained for CeHsp70-1 were 25 nmol/min/mg, 50 μM and 0.28 min⁻¹, respectively. The catalytic constant (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. This work provides a platform for kinetic studies of other members of the C. elegans Hsp70 molecular chaperones.
Department of Chemistry, Stephen F. Austin State University, Box 13006, SFA Station, Nacogdoches, TX 75904, USA. odunugaoo@sfasu.edu
Odutayo O
OO
Odunuga
Sarah A
SA
Bollinger
Ka H
KH
Choi
Elton I
EI
Polvadore
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D018840:HSP70 Heat-Shock Proteins; D011994:Recombinant Proteins; C081884:heat-shock protein 70.1; D000251:Adenosine Triphosphatases