Expression and sulfogalactolipid binding specificity of the recombinant testis-specific cognate heat shock protein 70

Abstract

Immunofluorescent studies with anti-2A antisera, raised specifically against a synthetic C-terminal peptide of native murine P70, the testes-specific cognate heat shock protein 70, demonstrated that the rat homologue of P70 is expressed on the surface of testicular cells. The murine hsp 70.2 gene, encoding P70, was cloned and expressed in Escherichia coli. The recombinant P70 (rP70) protein with a 6Xhistidine affinity tag at its amino terminus was purified from E. coli via nickel affinity column chromatography. Monoclonal anti-hsp70 antisera and anti-2A antisera cross-reacted with purified rP70. Binding of rP70 was specific for sulfogalactosylceramide (SGC) and sulfogalactosyglycerolipid (SGG). Binding was not inhibited by the sugar, galactose 3′ sulfate, nor was binding observed to desulfated derivatives of SGC and SGG, to other negatively charged lipids or other sulfated lipids. Furthermore, rP70 bound to an SGC-column and was eluted only at high salt in combination with high pH. These results show rP70 to possess a specific sulfatide binding site. Since the biochemical properties and immunoreactivity of rP70 are indistinguishable from native P70 and SLIP1 (testicular sulfoglycolipid immobilized protein 1) rP70 can be employed to examine the role of hsp70-mediated sulfatide binding in fertilization. Abbreviations: PAGE, polyacrylamide gel electrophoresis; IPTG, isopropylthio-β-D-galactoside; X-gal, 5-bromo-4-chloro-3-indolyl-β-D-galactoside; km, kanamycin; amp, ampicillin; kDa, kilodaltons; kb, kilobases; BHI, brain heart infusion; OPD, O-phenylenediamine dichloride peroxidase substrate; RT, room temperature; h, hours