The mammalian cytidine deaminases provide protection against viral invasion by deaminating cytidine, converting it to uracil. However, HIV-1 bypasses this enzyme by encoding in its genome the 23kDa protein Vif (Virulance Infectivity Factor), which targets Apobec3G for ubiquitination and degradation in the proteosome pathway. Virions without Apobec3G thereby can evade the host immunity and become infectious. It is our goal to investigate the interaction between the host deaminase and the viral Vif. The genes for three homologous deaminases (Apobec3G, AID, Apobec1), one antagonist (Vif) and one agonist (ACF) were obtained from outside sources and put into various vectors for expression trials in several bacterial cell lines. Vectors tried so far include untagged (i.e. His8-tagged) and tagged (Thioredoxin, Gluthatione-S-Transferase, and Maltose-Binding-Protein) proteins. Untagged and thioredoxin-tagged proteins were found to be expressed in inclusion bodies. GST-tagged proteins were successfully expressed in the soluble fraction, but difficulties were experienced with either cleavage or elution from glutathione resin. MBP-tagged proteins were also successfully expressed in the soluble fraction. Purification of MBP-Apobec1 was successful but Apobec1 becomes insoluble once cleaved from the fusion protein. Optimization of the purification protocol and refolding efforts are underway.
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