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Volume 272, Number 4, Issue of January 24, 1997 pp. 2578-2582
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Human B-Crystallin
SMALL HEAT SHOCK PROTEIN AND MOLECULAR CHAPERONE

(Received for publication, September 20, 1996, and in revised form, November 6, 1996)

Paul J. Muchowski , James A. Bassuk , Nicolette H. Lubsen ^§ and John I. Clark ^¶

From the Departments of  Biological Structure and ^¶ Ophthalmology, University of Washington, Seattle, Washington 98195-7420 and ^§ Department of Molecular Biology, University of Nijmegen, Toernooiveld, Nijmegen 6525 ED, The Netherlands

The polymerase chain reaction was used to amplify a cDNA sequence encoding the human B-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 B-crystallin was separated from MBP and Factor Xa by anion exchange chromatography. Recombinant B-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 (M_r ~22,000) that corresponded to total native human -crystallin and was recognized on Western immunoblots by antiserum raised against human B-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 B-crystallin and MBP-B fusion protein associated to form high molecular weight complexes that displayed chaperone-like function by inhibiting the aggregation of alcohol dehydrogenase at 37 °C and demonstrated the importance of the C-terminal domain of B-crystallin for chaperone-like activity.

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