Functional analysis of a small heat shock/α‐crystallin protein from Artemia franciscana

Oviparously developing embryos of the brine shrimp, Artemia franciscana , synthesize abundant quantities of a small heat shock/α‐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/α‐crystallin proteins.