Human αB-Crystallin: SMALL HEAT SHOCK PROTEIN AND MOLECULAR CHAPERONE

Paul J Muchowski,James A Bassuk,Nicolette H Lubsen,John I Clark

doi:10.1074/jbc.272.4.2578

Abstract

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.

Highlights

The polymerase chain reaction was used to amplify a cDNA sequence encoding the human ␣B-crystallin
Treatment of the affinity-purified fusion protein (Fig. 1A, lane 3) with the serine protease Factor Xa demonstrated that this fusion protein was cleaved into two distinct polypeptides (Fig. 1A, lane 4) that migrate to molecular weights corresponding to native maltose-binding protein (MBP) (ϳ43,000) and native human ␣B-crystallin (ϳ22,000)
Chemical Sequencing Results of Human ␣B-Crystallin—To verify that the Mr ϳ22,000 band corresponded to human ␣Bcrystallin, the cleaved fusion protein mixture was immobilized on a polyvinylidene difluoride membrane, and the Mr ϳ22,000 band was subjected to sequential Edman degradation for 15 cycles

Summary

SMALL HEAT SHOCK PROTEIN AND MOLECULAR CHAPERONE*

(Received for publication, September 20, 1996, and in revised form, November 6, 1996). 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 chaperonelike 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. The chemical nature of the interactions between human ␣-crystallins and other proteins is poorly understood because of the difficulty with isolation of sufficient quantities of unmodified protein from human lenses For this reason, recombinant techniques have been used to characterize structure-function relationships of the individual ␣-crystallin subunits. The recombinant expression system described here provided an excellent source of unmodified human ␣B-crystallin that assembled into a high molecular weight oligomer, and displayed chaperone-like activity against protein aggregation

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION