Figure 3

Purification and biochemical characterization of recombinantly expressed subtype-C Tat protein: (A) SDS-PAGE and Western blot analyses of the purified Tat protein. E. coli cells expressing Tat were harvested by centrifugation and lysed by sonication. Bacterial lysate was subjected to two successive strategies of protein purification, Ni-NTA and SP-Sepharose chromatographies. Measured quantity of the protein from different elutes was resolved on a 15% SDS-PAGE gel, M, protein molecular weight standards (# M3913, Sigma, St. Louis, Missouri, USA). Bottom pannel shows Western blot analysis of the Tat protein resolved on a duplicate SDS-PAGE gel and electrophoretically transferred to a PVDF membrane. A Tat-specific monoclonal antibody (# 4138, NIH AIDS Research and Reference Reagent Program) was used for the Western blot. (B) MALDI-TOF spectrum of the purified Tat protein. Purified and lyophilized Tat protein was reconstituted in sterile distilled water and subjected to MALDI-TOF analysis. (C) CD spectra of B-Tat and C-Tat were measured from 250 to 190 nm with a 0.1 cm path length in 10 mM phosphate buffer (pH 7.0).