Autoprocessing of the HIV‐1 protease using purified wild‐type and mutated fusion proteins expressed at high levels in Escherichia coli

Abstract

Various constructs of the human immunodeficiency virus, type 1 (HIV-1) protease containing flanking Pol region sequences were expressed as fusion proteins with the maltose-binding protein of the malE gene of Escherichia coli. The full-length fusion proteins did not exhibit self-processing in E. coli, thereby allowing rapid purification by affinity chromatography on cross-linked amylose columns. Denaturation of the fusion protein in 5 M urea, followed by renaturation, resulted in efficient site-specific autoprocessing to release the 11-kDa protease. Rapid purification involving two column steps gave an HIV-1 protease preparation of > 95% purity (specific activity z 8500 pmol . min-' . pg protease-') with an overall yield of about 1 mg/l culture. Incubation of an inactive mutant protease fusion protein with the purified wild-type protease resulted in specific trans cleavage and release of the mutant protease. Analysis of products of the HIV-1 fusion proteins containing mutations at either the Nor the C-terminal protease cleavage sites indicated that blocking one of the cleavage sites influences the cleavage at the non-mutated site. Such mutated full-length and truncated protease fusion proteins possess very low levels of proteolytic activity ( z 5 pmol . min-l . pg protein-').The human immunodeficiency virus (HIV) is the causative agent of the acquired immunodeficiency syndrome (AIDS) [l, 21. One of the potential targets for the inhibition of virus maturation is the virally encoded protease which is responsible for the processing of the precursor polyproteins into the necessary structural proteins and replication enzymes [3, 41. Recently, it was demonstrated that inhibitors of the HIV-1 protease arrest the maturation of HIV-I-like particles [5, 61. Structural analysis of the active HIV-1 protease has shown that it is a homodimer possessing one active site. The active site contains two sequences of AspThrGly, in common with the other known aspartic acid proteases [7, 81. Active-site mutation (Asp25) of the protease resulted in the production of non-infectious progeny virus [9].Although dimerization of the protease in the polyprotein form is clearly a prerequisite for activity [7, 81, there is no knowledge on the mechanism of activation of the protease from the Gag-Pol polyprotein and the order of the various cleavages that occur in the polyprotein. In order to study the mechanism of HIV-1 protease action in vitro, we developed a system to produce large quantities of purified HIV-1 protease. Others have expressed the protease gene (297 bp) in Escherichia coli, but with very low yield [lo]. The protease gene expressed as a fusion with the flanking truncated gag-pol region sequences resulted in the self-processing of the protease in E. coli and purification involved several successive steps resulting again in low recovery of pure protease [ll].Here we describe the expression of the HIV-1 protease gene containing a portion of the flanking pol gene in fusion with the malE gene of E. coli which codes for the maltose ...

Construction of an Expression System for Aqualysin I inEscherichia coliThat Gives a Markedly Improved Yield of the Enzyme Protein

“…Louis et al reported that a fusion protein of human immunodeficiency virus type 1 protease (HIV-1) with MBP was not self-processed in E. coli cells, and Wan et al indicated that recombinant HIV-1 expressed as fusion proteins might affect the dimerization of this enzyme which is necessary to the formation of the protease active site, resulting in a reduction in toxicity to the host cells. 10,11) These results are consistent with the present observation that the fused proteins appeared to suppress effectively the folding-promoting activity of the Nterminal propeptide within the aqualysin-precursor, leading to greatly improved recovery of the mature enzyme.…”

“…Louis et al have reported that selfprocessing of immunodeficiency virus type 1 protease (HIV-1) in E. coli was suppressed by fusing the protein with MBP. 10) We constructed fusion aqualysin I expression plasmids in which the signal peptide portion of the AQNÁC105 gene was replaced with a coding sequence of maltose binding protein (MBP), glutathione S-transferase (GST), or thioredoxin (Trx). When these fusion genes were introduced and were allowed to be expressed in E. coli, only a very low level of protease activity was detected in the crude extracts, but the activity was greatly enhanced on heat treatment of the extract at 70 C ( Table 1).…”

Human immunodeficiency virus type 1 viral background plays a major role in development of resistance to protease inhibitors.

“…HIV-1 NL4-3 protease genes containing the amino acid substitutions were generated as described (25). The mutant protease genes were cloned into the pMAL-P2 expression vector and expressed in Escherichia coli, and the fusion protein was purified as reported (29), with the exception of allowing the combination mutant proteases to autoprocess for longer times (16-48 h) before FPLC Mono S chromatography.…”

Studies on the Symmetry and Sequence Context Dependence of the HIV-1 Proteinase Specificity

“…Enzyme Assay-Purified HIV-1 proteinase was prepared as described previously (16). Active site titration for the HIV-1 proteinase was performed with compound 3 (17).…”