Large Scale Purification and Refolding of HIV-1 Protease from Escherichia Coli Inclusion Bodies

Overview

Journal J Protein Chem

Specialties Biochemistry
Chemistry

Date 1993 Jun 1

PMID 8397790

Citations 24

Authors

J O Hui

A G Tomasselli

I M Reardon

J M Lull

D P Brunner

C S Tomich

R L Heinrikson

Affiliations

Soon will be listed here.

Abstract

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.

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