Human alphaB-crystallin. Small heat shock protein and molecular chaperone.
The polymerase chain reaction was used to amplify a cDNA sequence encoding the human alphaB-crystallin. The amplified cDNA fragment was cloned into the bacterial expression vector pMAL-c2 and expressed as a soluble fusion protein coupled to maltose-binding protein (MBP). After maltose affinity chromatography and cleavage from MBP by Factor Xa, the recombinant human alphaB-crystallin was separated from MBP and Factor Xa by anion exchange chromatography. Recombinant alphaB-crystallin was characterized by SDS-polyacrylamide electrophoresis (PAGE), Western immunoblot analysis, Edman degradation, circular dichroism spectroscopy, and size exclusion chromatography. The purified crystallin migrated on SDS-PAGE to an apparent molecular weight (Mr approximately 22,000) that corresponded to total native human alpha-crystallin and was recognized on Western immunoblots by antiserum raised against human alphaB-crystallin purified from lens homogenates. Chemical sequencing, circular dichroism spectroscopy, and size exclusion chromatography demonstrated that the recombinant crystallin had properties similar or identical to its native counterpart. Both recombinant alphaB-crystallin and MBP-alphaB fusion protein associated to form high molecular weight complexes that displayed chaperone-like function by inhibiting the aggregation of alcohol dehydrogenase at 37 degrees C and demonstrated the importance of the C-terminal domain of alphaB-crystallin for chaperone-like activity.
Muchowski
P J
PJ
Department of Biological Structure, University of Washington, Seattle, Washington 98195-7420, USA.
Bassuk
J A
JA
Lubsen
N H
NH
Clark
J I
JI
D015153:Blotting, Western; D003001:Cloning, Molecular; D003459:Crystallins / Q000737:chemistry*; D018076:DNA, Complementary / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D007908:Lens, Crystalline / Q000737:chemistry; D018832:Molecular Chaperones / Q000737:chemistry*; D008970:Molecular Weight
D003459:Crystallins; D018076:DNA, Complementary; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones