Rapid purification and characterisation of HIV‐1 reverse transcriptase and RNaseH engineered to incorporate a C‐terminal tripeptide α‐tubulin epitope

Abstract

HIV reverse rr#nscripcvaie; RNuLEW; Probin cnpinecrin$: s-Trrbulin epiropc; Immunaulfniry puriReatian I. INTRODUCTIONReverse transcriptase is an enzyme of central importancc in rhc replication of HIV, the causative agent of AIDS [l]. Part of the HIV-I polgenecodes for a p66RT subunit that is cleaved at the C-terminal region by the HIV protcase to give a p66/p51 heterodimcr, the form of RT observed in virions f2), An RNaseH domain has been identified within the last 120 amino acids of the Cterminal region of the p66 subunit by sequence analysis [3] and functional studies [4].We have previously described immunoaffinity chromatography of RT using immobilized monoclonal antibodies raised to HIV-I RT [5]. Elution of RT from these columns requires vigorous conditions which may have deleterious effects on enzyme structure. This together with the C-terminal heterogeneity observed in these preparations [6], may contribute to the disorder observed in three different crystal forms of RT when ex- amincd in an X-ray beam [7]. We thus wished to devise a mild isolation procedure which could produce protein with an intact C-terminus, The work of Wehland et al, [8] on the detailed mapping of the amino acid sequence requirement for the binding of m-tubulin to the monoclonal antibody YL1/2 [9] showed that an essentially linear epitope was located at the C-terminus OF the polypeptide chain. There was a requirement for a C-terminal aromatic residue (either tyrosine or phenylalanine) containing a free carboxyl group, preceded by an acidic residue (glutamic or aspartic acid). A further adjacent acidic residue greatly increased binding, It was also demonstrated that the loss of the C-terminal aromatic residue abolished the binding of cr-tubulin as well as related peptides to the YL1/2 antibody [8]. We therefore reasoned that incorporation of this epitope into HIV RT and RNaseH could afford a method of purifying these proteins with a homogeneous C-terminus using a simple peptide elution procedure from columns of immobilized YLl/:! antibody. Incorporation of this a-tubulin epitope into p66RT and RNaseH can be achieved by mutating the C-terminus from Lys-Val-Leu to Glu-Glu-Phe. The site of HIV protease cleavage of the p66RT subunit has been shown to be between a Phe and Tyr giving a ~51 subunit with a C-terminal sequence of Glu-Thr+he [IO]. Therefore only a single change of Thr to Glu is required to incorporate the C+ tubulin epitope at the C-terminus of the ~51 polypeptide. In changing the natural amino acid sequence of a protein there is the possibility of changing the biological characteristics of that protein. It has, however, previously been shown that the C-terminus of HIV-1 RT 298Published by Elsevier Sciettce Publishers B. V.

Inhibition of Human Cytomegalovirus DNA Polymerase by C-Terminal Peptides from the UL54 Subunit

“…Following further washes, the wells were incubated with monoclonal antibody (MAb) YL1/2 (diluted 1:50 in PBS containing 2% FCS) for 1 h at 37°C. MAb YL1/2, which recognizes the EEF epitope inserted at the C terminus of UL44 (46), was purchased from Serotec Ltd. Plates were then washed and incubated with horseradish peroxidase (HRP)-conjugated anti-rat antibody (Sigma) (diluted 1:500 in PBS containing 2% FCS). After the washes, the chromogenic substrate 2,2Ј-azino-bis(3-ethylbenzthiazinzoline-6-sulfonic acid (ABTS; Dynatech) in citrate phosphate buffer (pH 4.0) containing 0.01% hydrogen peroxide was added, and absorbance was read at 405 nm on a Multiskan plate reader (Titertek, ICN Biomedicals).…”

Cloning and characterization of E2F-2, a novel protein with the biochemical properties of transcription factor E2F.

“…The 3' primer used (5'-GAGAGCAAGCTlTAG AACTCCTCATTAATCAACAGGTCCCCAAGG-3') added three amino acids (Glu-Glu-Phe) to the natural C terminus of the protein for antibody recognition by the YL1/2 antitubulin antibody (Harlan Bioproducts, Indianapolis, Ind.) (41) and also included a HindIII site. The DNA was cloned as a BamHI-HindIII fragment into pT5T digested with BamHI and HindIII to generate pE2F-2-PCR.…”

“…Full-length E2F-2 protein with the C-terminal tubulin tripeptide epitope was affinity purified essentially as described previously (41) and electroeluted from an SDS-polyacrylamide gel as previously described (25). The conditions for the gel shift assay, the affinity purification of HeLa E2F (25), and the purification of recombinant pRb6O (14) have been previously described.…”

A Comparative Study of the in vitro and in vivo Antiviral Activities of Acyclovir and Penciclovir

“…VZV DNA polymerase was co-expressed with its accessory protein (gene 16) in insect cells using a recombinant baculovirus expression system (Ertl et el., 1991; C. Ohmstede and S. Short, Burroughs Wellcome Co., NC 27709, unpublished results). The accessory protein was expressed with the threeamino-acid motif (EEF) at the Cterminus, which forms the recognition site for the monoclonal antibody YL1/2 (Stammers et et., 1991). This was used for immunoaffinity purification of the complex similarly to that described by Ertl and Powell (1992).…”