Characterization, cloning, and expression of porcine αB crystallin

α-Crystallin is a major lens protein present in the lenses of all vertebrate species. Recent studies have revealed that bovine α-crystallins possess genuine chaperone activity similar to small heat-shock proteins. In order to compare this chaperone-like structural protein from the eye lenses of different mammalian species, we have cloned and expressed one of the main α-crystallin subunits, i.e., αβ crystallin, from the porcine lenses in order to facilitate the structure-function evaluation and comparison of this chaperonin protein. cDNA encoding αβ subunit chain was obtained using a new 'Marathon cDNA amplification' protocol of Polymerase Chain Reaction (PCR). PCR-amplified product corresponding to αβ subunit was then ligated into pGEM-T plasmid and prepared for nucleotide sequencing by the dideoxynucleotide chain-termination method. Sequencing several positive clones containing DNA inserts coding for αβ-crystallin subunit constructed only one complete full-length reading frame of 525 base pairs similar to human and bovine αβ subunits, covering a deduced protein sequence of 175 amino acids including the universal translation-initiating methionine. The porcine αβ crystallin shows only 3 and 7 residues difference to bovine and human αβ crystallins respectively, revealing the close relatedness among mammalian eye lens proteins. The sequence differences between porcine and submammalian species such as chicken and bullfrog are much greater, especially at the N- and C-terminal regions of these αβ crystallins. Expression of αβ subunit chain in E. coli vector generated a polypeptide which can cross-react with the antiserum against the native and purified αβ subunit from the native porcine lenses albeit with a much lower activity.