Large scale purification and refolding of HIV-1 protease fromEscherichia coli inclusion bodies |
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Authors: | John O Hui Alfredo G Tomasselli Ilene M Reardon June M Lull David P Brunner Che-Shen C Tomich and Robert L Heinrikson |
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Institution: | (1) The Upjohn Company, 301 Henrietta Street, 49001 Kalamazoo, Michigan |
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Abstract: | The protease encoded by the human immunodeficiency virus type 1 (HIV-1) was engineered inEscherichia coli as a construct in which the natural 99-residue polypeptide was preceded by an NH2-terminal methionine initiator. Inclusion bodies harboring the recombinant HIV-I protease were 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 second of two peaks, migrated in SDS-PAGE as a single sharp band ofM
r 10,000. The purified HIV-1 protease was refolded into an active enzyme by diluting a solution of the protein in 50% acetic acid with 25 volumes of buffer atpH 5.5. This method of purification, which has also been applied to the purification of HIV-2 protease, provides a single-step procedure to produce 100 mg quantities of fully active enzyme. |
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Keywords: | HIV-1 protease gel-filtration Superdex 75 FPLC column reversed-phase HPLC |
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