PURIFICATION OF RECOMBINANT HUMAN HSP60 - USE OF A GROEL-FREE PREPARATION TO ASSESS AUTOIMMUNITY IN RHEUMATOID-ARTHRITIS

A 65 kDa mycobacterial heat shock protein has been implicated in the d evelopment or perpetuation of the inflammatory diseases rheumatoid art hritis (RA) and insulin dependent diabetes mellitus (IDDM). An homolog y of the mycobacterial hsp65 with human hsp60 (HuHsp60) has been thoug ht to constitute a cross reactive autoimmunizing pathogenetic potentia l. Study of this cross reactivity with recombinant reagents has been c omplicated by the fact that recombinant HuHsp60 might be contaminated by the E. coli homologue of HuHsp60, groEL. GroEL and HuHsp60 are very similar in isoelectric point and molecular weight and therefore diffi cult to separate by classical physicochemical means, Therefore, the Hu Hsp60 gene was subcloned into the vector, pRSET-B, which resulted in r ecombinant HuHsp60 protein fused to a 4.5 kDa peptide containing a pol yhistidine hexamer. Metal ion affinity for the polyhistidine allowed t he rapid and efficient chromatographic separation of the HuHsp60 from groEL. Rabbit antisera were developed to linear peptide epitopes uniqu e to either HuHsp60 or groEL and utilized to discriminate between thes e proteins during their separation. With the newly prepared HuHsp60 we show that the amount of anti-HuHsp60 autoantibody in both RA and norm al sera was too great to be accounted for by cross reacting anti-MbHsp 65. (C) 1995 Academic Press Limited