Functional analysis of a small heat shock/alpha-crystallin protein from Artemia franciscana. Oligomerization and thermotolerance.
Oviparously developing embryos of the brine shrimp, Artemia franciscana, synthesize abundant quantities of a small heat shock/alpha-crystallin protein, termed p26. Wild-type p26 functions as a molecular chaperone in vitro and is thought to help encysted Artemia embryos survive severe physiological stress encountered during diapause and anoxia. Full-length and truncated p26 cDNA derivatives were generated by PCR amplification of p26-3-6-3, then cloned in either pET21(+) or pRSETC and expressed in Escherichia coli BL21(DE3). All constructs gave a polypeptide detectable on Western blots with either p26 specific antibody, or with antibody to the His(6) epitope tag encoded by pRSETC. Full-length p26 in cell-free extracts of E. coli was about equal in mass to that found in Artemia embryos, but p26 lacking N- and C-terminal residues remained either as monomers or small multimers. All p26 constructs conferred thermotolerance on transformed E. coli, although not all formed oligomers, and cells expressing N-terminal truncated derivatives of p26 were more heat resistant than bacteria expressing p26 with C-terminal deletions. The C-terminal extension of p26 is seemingly more important for thermotolerance than is the N-terminus, and p26 protects E. coli against heat shock when oligomer size and protein concentration are low. The findings have important implications for understanding the functional mechanisms of small heat shock/alpha-crystallin proteins.
Crack
Julie A
JA
Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada.
Mansour
Marc
M
Sun
Yu
Y
MacRae
Thomas H
TH
D000818:Animals; D001156:Artemia / Q000737:chemistry*; D002498:Centrifugation / Q000379:methods; D003001:Cloning, Molecular; D003459:Crystallins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology; D018869:Heat-Shock Response; D016296:Mutagenesis; D012995:Solubility
D003459:Crystallins