Functional analysis of a small heat shock/alpha-crystallin protein from Artemia franciscana. Oligomerization and thermotolerance.

Abstract

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.

• Authors: Julie A Crack, Marc Mansour, Yu Sun, Thomas H MacRae
• Journal: European journal of biochemistry (Eur J Biochem) Vol. 269 Issue 3 Pg. 933-42 (Feb 2002) ISSN: 0014-2956 [Print] England
• PMID: 11846795 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Crystallins

Topics

• Animals
• Artemia (chemistry)
• Centrifugation (methods)
• Cloning, Molecular
• Crystallins (chemistry, genetics, metabolism)
• Escherichia coli (genetics, physiology)
• Heat-Shock Response
• Mutagenesis
• Solubility