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Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Poster Abstracts
79
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.