Biophysical Society Thematic Meeting - November 16-20, 2015

Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Poster Abstracts

47-POS Board 47 Novel Expression System for HIV-1 Subtype C Protease Alison Williams , Ikechukwu Achilonu, Yasien Sayed. University of Witwatersrand, Johannesburg, 2050, South Africa.

HIV-1 is the most common form of HIV and can be subdivided into groups and subtypes. The subtype of interest for this study, subtype C, can be found in southern Africa. HIV-1 protease is the main drug target and belongs to the class of aspartic proteases. The aim of this study was to develop a novel purification system using a thioredoxin His-tagged fusion protein to improve the protease yield. The study looked at a clinical variant (designated L38↑N↑L) and wild-type protease. The L38↑N↑L variant was found in a drug naïve infant whose mother was exposed to ARV therapy as part of the prevention of mother-to-child transmission (PMTCT) initiative. This double of Asn and Leu results in a protease with each subunit containing 101 amino acids rather than 99. The wild-type and variant proteins were successfully overexpressed in BL21 (DE3) pLysS E.coli cells and purified using nickel charged IMAC. The secondary structure was characterised using far-UV circular dichroism and consisted of β-sheets. A cleavage assay was conducted using a fluorogenic substrate and both the wild-type and the variant were found to be active. The percentage active sites were determined using isothermal titration calorimetry and were found to be 13% for wild-type protease and 8% for the L38↑N↑L variant. Subsequently, the quaternary structure was characterised using size exclusion high performance liquid chromatography wild-type protease was found to be predominately monomeric and the L38↑N↑L variant was found to be dimeric.

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