LARGE-SCALE PURIFICATION AND REFOLDING OF HIV-1 PROTEASE FROM ESCHERICHIA-COLI INCLUSION-BODIES

The protease encoded by the human immunodeficiency virus type 1 (HIV-1 ) was engineered in Escherichia coli as a construct in which the natur al 99-residue polypeptide was preceded by an NH2-terminal methionine i nitiator. Inclusion bodies harboring the recombinant HIV-1 protease we re dissolved in 50% acetic acid and the solution was subjected to gel filtration on a column of Sephadex G-75. The protein, eluted in the se cond of two peaks, migrated in SDS-PAGE as a single sharp band of M(r) almost-equal-to 1 0,000. The purified HIV-1 protease was refolded int o an active enzyme by diluting a solution of the protein in 50% acetic acid with 25 volumes of buffer at pH 5.5. This method of purification , which has also been applied to the purification of HIV-2 protease, p rovides a single-step procedure to produce 100 mg quantities of fully active enzyme.