中国生物工程杂志

Inducible heat shock protein 70 (HSP70) is a major heat shock protein induced under stress conditions and functions mainly as chaperone. HSP70 molecule consists of two domains, N-terminal ATP binding domain(ATPBD) and C-terminal polypeptide binding domains(PBD). A nuclear localization signal(NLS) is within ATP binding domain(ATPBD). The DNA-coding regions of full-length HSP70(HSP70wt), HSP70ΔATPBD, HSP70ΔPBD and HSP70ΔNLS were cloned into a bacterial expression vector pQE-31, respectively, by PCR. HSP70 and its truncated mutants were over-expressed in Escherichia coli M15 and the fusion proteins were purified with Ni2+chelating affinity column and further purified by DEAE-cellulose to apparent homogeneity. Based on SDS-PAGE, both HSP70 and HSP70ΔNLS were 70 kDa, HSP70ΔPPBD 52 kDa and HSP70ΔATPBD 28 kDa. HSP70, HSPΔATPBD, HSP70ΔPPBD and HSP70ΔNLS that had been electrophoresized on SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (PVDF) were all recognized on Western immunoblot by anti-human HSP70 antibody. In PBS buffer, HSP70wt, HSPΔATPBD, HSP70ΔPPBD and HSP70ΔNLS were all proved to exist in monomers using gel filtration chromatography. In order to check whether the recombinant proteins can interact with nucleolin (C23) , a protein dominantly distributed in nucleolus. The purified proteins that had been fractioned on SDS-PAGE and transferred to PVDF were first incubated with nuclear extracts containing nucleolin and then visualized by anti- nucleolin antibody (protein overlay assay). The in vitro protein overlay assay showed that HSP70 and HSP70ΔNLS and HSPΔATPBD can interact with nucleolin, while HSP70ΔPPBD can not, suggesting that the C-terminal polypeptide binding domain of HSP70 is sufficient for the interaction between HSP70 and nucleolin.