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-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 alpha-crystallin subunits, i.e., alpha B crystallin, from the porcine lenses in order to facilitate the structure-function evaluation and comparison of this chaperonin protein. cDNA encoding alpha B subunit chain was obtained using a new "Marathon cDNA amplification" protocol of Polymerase Chain Reaction (PCR). PCR-amplified product corresponding to alpha B subunit was then ligated into pGEM-T plasmid and prepared for nucleotide sequencing by the dideoxy-nucleotide chain-termination method. Sequencing several positive clones containing DNA inserts coding for alpha B-crystallin subunit constructed only one complete full-length reading frame of 525 base pairs similar to human and bovine alpha B subunits, covering a deduced protein sequence of 175 amino acids including the universal translation-initiating methionine. The porcine alpha B crystallin shows only 3 and 7 residues difference to bovine and human alpha B crystallins respectively, revealing the close relatedness among mammalian eye lens proteins. The sequence differences between porcine and sub-mammalian species such as chicken and bullfrog are much greater, especially at the N- and C-terminal regions of these alpha B crystallins. Expression of alpha B subunit chain in E. coli vector generated a polypeptide which can cross-react with the antiserum against the native and purified alpha B subunit from the native porcine lenses albeit with a much lower activity.
Liao
J H
JH
Laboratory of Crystallin Research, National Taiwan University, Taipei.
Hung
C C
CC
Lee
J S
JS
Wu
S H
SH
Chiou
S H
SH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002645:Chickens; D003001:Cloning, Molecular / Q000379:methods; D003459:Crystallins / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D018076:DNA, Complementary / Q000302:isolation & purification; D004284:Dogfish; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011892:Rana catesbeiana; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013552:Swine / Q000235:genetics / Q000378:metabolism*
D003459:Crystallins; D018076:DNA, Complementary; D018832:Molecular Chaperones; D011994:Recombinant Proteins