Aids Related Proteins--Structure And Function

Diabetes, Digestive, Kidney Diseases

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Abstract

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. These proteins have since been successfully produced in Hi5 insect cells using a baculovirus vector. Soluble protein of Apobec3G was obtained in sufficient quantity for crystallization trials, which produced tiny crystals under several conditions and attempts have been made to obtain usable crystals for X-ray diffraction.