Cheng Y S, McGowan M H, Kettner C A, Schloss J V, Erickson-Viitanen S, Yin F H
Central Research and Development Department, E.I. duPont de Nemours and Co., Wilmington, DE 19880-0328.
Gene. 1990 Mar 15;87(2):243-8. doi: 10.1016/0378-1119(90)90308-e.
A complete chemical synthesis and assembly of genes for the production of human immunodeficiency virus type-I protease (HIV-PR) and its precursors are described. The T7 expression system was used to produce high levels of active HIV-PR and its precursors in Escherichia coli inclusion bodies. The gene encoding the open reading frames of HIV-PR was expressed in E. coli as a 10-kDa protein, while the genes encoding HIV-PR precursors were expressed as larger proteins, which were partially processed in E. coli to the 10-kDa form. These processing events are autoproteolytic, since a single-base mutation, changing the active-site aspartic acid to glycine, completely abolished the conversion. HIV-PR can be released with 8 M urea from washed cellular inclusion bodies, resulting in a preparation with few bacterial host proteins. After refolding, this preparation contains no nonspecific protease or peptidase activities. The recombinant HIV-PR isolated from inclusion bodies cleaves HIV-PR substrates specifically with a specific activity comparable to column-purified HIV-PR.
