Jo. Hui et al., LARGE-SCALE PURIFICATION AND REFOLDING OF HIV-1 PROTEASE FROM ESCHERICHIA-COLI INCLUSION-BODIES, Journal of protein chemistry, 12(3), 1993, pp. 323-327
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.