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 natural 99-residue polypeptide was preceded by an NH2-terminal methionine initiator. Inclusion bodies harboring the recombinant HIV-1 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 of M(r) approximately 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 at pH 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.
Upjohn Company, Kalamazoo, Michigan 49001.
J O
JO
Hui
A G
AG
Tomasselli
I M
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Reardon
J M
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Lull
D P
DP
Brunner
C S
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Tomich
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Heinrikson
D000085:Acetates; D019342:Acetic Acid; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology*; D016333:HIV Protease / Q000737:chemistry / Q000302:isolation & purification*; D015497:HIV-1 / Q000201:enzymology; D002479:Inclusion Bodies / Q000201:enzymology*; D008969:Molecular Sequence Data; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D014764:Viral Proteins / Q000737:chemistry / Q000302:isolation & purification*
D000085:Acetates; D011994:Recombinant Proteins; D014764:Viral Proteins; D016333:HIV Protease; D019342:Acetic Acid