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 development or perpetuation of the inflammatory diseases rheumatoid arthritis (RA) and insulin dependent diabetes mellitus (IDDM). An homology of the mycobacterial hsp65 with human hsp60 (HuHsp60) has been thought to constitute a cross reactive autoimmunizing pathogenetic potential. Study of this cross reactivity with recombinant reagents has been complicated 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 difficult to separate by classical physicochemical means. Therefore, the HuHsp60 gene was subcloned into the vector, pRSET-B, which resulted in recombinant HuHsp60 protein fused to a 4.5 kDa peptide containing a polyhistidine hexamer. Metal ion affinity for the polyhistidine allowed the rapid and efficient chromatographic separation of the HuHsp60 from groEI. Rabbit antisera were developed to linear peptide epitopes unique to either HuHsp60 or groEL and utilized to discriminate between these proteins during their separation. With the newly prepared HuHsp60 we show that the amount of anti-HuHsp60 autoantibody in both RA and normal sera was too great to be accounted for by cross reacting anti-MbHsp65.
Handley
H H
HH
Sam and Rose Stein Institute for Research on Aging, San Diego, La Jolla, CA, USA.
Ngyuen
M D
MD
Yu
D T
DT
Gupta
R S
RS
Vaughan
J H
JH
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000032:analysis; D001172:Arthritis, Rheumatoid / Q000276:immunology*; D001323:Autoantibodies / Q000032:analysis; D001483:Base Sequence; D018834:Chaperonin 60 / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D013045:Species Specificity
D000907:Antibodies, Bacterial; D001323:Autoantibodies; D018834:Chaperonin 60; D007074:Immunoglobulin G; D011994:Recombinant Proteins