CHARACTERIZATION, CLONING, AND EXPRESSION OF PORCINE ALPHA-B-CRYSTALLIN

alpha-Crystallin is a major lens protein present in the lenses of all vertebrate species. Recent studies have revealed that bovine alpha-cry stallins possess genuine chaperone activity similar to small heat-shoc k proteins. In order to compare this chaperone-like structural protein from the eye lenses of different mammalian species, we have cloned an d expressed one of the main alpha-crystallin subunits, i.e., alpha bet a crystallin, from the porcine lenses in order to facilitate the struc ture-function evaluation and comparison of this chaperonin protein. cD NA encoding alpha beta subunit chain was obtained using a new ''Marath on cDNA amplification'' protocol of Polymerase Chain Reaction (PCR). P CR-amplified product corresponding to alpha beta subunit was then liga ted into pGEM-T plasmid and prepared for nucleotide sequencing by the dideoxynucleotide chain-termination method. Sequencing several positiv e clones containing DNA inserts coding for alpha beta-crystallin subun it constructed only one complete full-length reading frame of 525 base pairs similar to human and bovine alpha beta subunits, covering a ded uced protein sequence of 175 amino acids including the universal trans lation-initiating methionine. The porcine alpha beta crystallin shows only 3 and 7 residues difference to bovine and human alpha beta crysta llins respectively, revealing the close relatedness among mammalian ey e lens proteins. The sequence differences between porcine and submamma lian species such as chicken and bullfrog are much greater, especially at the N- and C-terminal regions of these alpha beta crystallins. Exp ression of alpha beta subunit chain in E. coli vector generated a poly peptide which can cross-react with the antiserum against the native an d purified alpha beta subunit from the native porcine lenses albeit wi th a much lower activity. (C) 1998 Academic Press.