An improved method for purification and refolding of recombinant HIV Vif expressed in Escherichia coli.
Virion infectivity factor (Vif) is a 23 kDa protein that protects HIV-1 from deamination of its proviral DNA by APOBEC3G. The active form of Vif is a multimer that interacts simultaneously with CBF-beta, the elongin B and C subunits, Cullin 5, and APOBEC3G to form a ubiquitin ligase complex targeting the latter for degradation. Vif clearly represents an attractive target for developing novel antiviral drugs for the therapy of HIV/AIDS, and this goal requires a source of well folded, readily available protein. For that purpose, we have cloned Vif in the pET28a expression vector, expressing the resulting His-tagged recombinant protein in the BL21(DE3) Escherichia coli strain. After lysis, Vif was solubilized from the insoluble fraction with 6 M guanidinium chloride and purified by denaturing immobilized-metal affinity chromatography, refolding the protein afterwards by dialysis. The use of 2-(N-morpholino)ethanesulfonic acid buffer at pH 6.2 and the presence of EDTA improved Vif refolding yields by reducing the formation of insoluble aggregates. The purified protein was bound by two monoclonal antibodies against sequential and conformational epitopes located at the C and N terminus, respectively.
Physical-Chemistry Division, Bioinformatics Department, Center for Genetic Engineering and Biotechnology, La Habana, Cuba.
Carlos A
CA
Duarte
Physical-Chemistry Division, Bioinformatics Department, Center for Genetic Engineering and Biotechnology, La Habana, Cuba.
Mickel
M
Palomino
D017738:Alkanesulfonic Acids / Q000737:chemistry; D002021:Buffers; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D009025:Morpholines / Q000737:chemistry; D066329:Protein Aggregates; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D054320:vif Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification
D017738:Alkanesulfonic Acids; D002021:Buffers; D009025:Morpholines; D066329:Protein Aggregates; D011994:Recombinant Proteins; D054320:vif Gene Products, Human Immunodeficiency Virus; C514847:vif protein, Human immunodeficiency virus 1; C004550:2-(N-morpholino)ethanesulfonic acid
EDTA; HIV; Vif; Zn binding motif; purification; refolding