Figure 4

Purification and biochemical characterization of recombinantly expressed subtype-B Tat protein: (A) SDS-PAGE and Western blot analyses of the purified Tat protein. Tat expression cassette was amplified from a standard HIV-1 subtype B molecular clone, YU-2, and subcloned into a bacterial expression vector. 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 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.