Development of a Novel HTLV-1 Protease: Human Fcγ1 Recombinant Fusion Molecule in the CHO Eukaryotic Expression System

Abstract

Human T-cell leukaemia virus type 1 (HTLV-1) is the causative agent of two life-threatening diseases, adult T cell leukaemia/lymphoma (ATLL), and HTLV-1-associated myelopathy/tropical spastic (HAM/TSP). HTLV-1 protease (HTLV-1-PR) is an aspartic protease that represents a promising target for therapeutic purposes like human immunodeficiency virus-PR inhibitors (HIV-PR). Therefore, in this study, the human Fc fusion recombinant-PR (HTLV-1-PR:hFcγ1) was designed and expressed for two applications, finding a blocking substrate as a potential therapeutic or a potential subunit peptide vaccine. The PCR amplified DNA sequences encoding the HTLV-1-PR from the MT2-cell line using specific primers with restriction enzyme sites of Not1 and Xba1. The construct was then cloned to pTZ57R/T TA plasmid and, after confirming the PR sequence, subcloned into the pDR2ΔEF1α Fc-expression vector to create pDR2ΔEF1α.HTLV-1-PR:hFcγ1. The integrity of recombinant DNA was confirmed by sequencing to ensure that the engineered construct was in the frame. The recombinant fusion protein was then produced in the Chinese hamster ovary cell (CHO) system and was purified from its supernatant using HiTrap-rPA column affinity chromatography. Then, the immunofluorescence assay (IFA) co-localisation method showed that HTLV-1-PR:hFc recombinant fusion protein has appropriate folding as it binds to the anti-Fcγ antibody; the Fcγ1 tag participates to have HTLV-1-PR:hFcγ1 as a dimeric secretory protein. The development and production of HTLV-1-PR can be used to find a blocking substrate as a potential therapeutic molecule and apply it in an animal model to assess its immunogenicity and potential protection against HTLV-1 infection.

Keywords: Adult T cell leukaemia/lymphoma (ATLL); Chinese hamster ovary cell (CHO) expression system; HTLV-1 protease; Human T-cell leukaemia virus type 1 (HTLV-1); Recombinant Fc-fusion protein.

Fig. 1

Bioinformatic analysis and homology modelling of HTLV-1-PR:hFcγ1 fusion protein. A Solid ribbon view of the modelled HTLV-1-PR:hFcγ1 dimer, positions of the disulphide bonds (Cys²⁶⁴-Cys³²⁴, Cys³⁷⁰-Cys⁴²⁸), and N-glycosylation sites: Asn¹⁶⁸ and Asn³⁰⁰. B Solvent surface with glycosylation sites of HTLV-1-PR:hFcγ1 dimer

Fig. 2

N-glycosylation analysis. The predicted N-glycosylation sites of the HTLV-1-PR:hFcγ1 fusion protein sequence were determined using the NetNGlyc 1.0 Server (https://services.healthtech.dtu.dk/service.php?NetNGlyc-1.0). The HTLV-1-PR:hFcγ1 contains four potential N-glycosylation sites

Fig. 3

The colony PCR of transformant cells. Five colonies were selected randomly and tested by PCR. The presence of a 512-bp fragment on 1.5% agarose gel confirms the appropriate gene transformation

Fig. 4

A Schematic image of pDR2DEF1a.HTLV-1-PR:hFcγ1 eukaryotic expression vector. B Agarose gel electrophoresis of the HTLV-1-PR product. Double-digestion expression vector, pDR2DEF1a.HTLV-1, and plasmid with HTLV-1. Lane L: 1-kb DNA size marker (Thermo Fisher Scientific, USA). (A) Undigested pTZ57R/T plasmid: a band of 3000 bp with incorporated sequences of two restriction sites, (B) digested plasmid: the HTLV-1-PR weight is in the 500 kb, (C) undigested expression vector, and (D) digested vector

Fig. 5

The nucleotide sequences of recombinant HTLV-1-PR:Fc fused into the C-terminal of Fcγ1 (hinge region, Cγ2 and Cγ3). The ExPASy Translate tool (http://www.expasy.org/tools/dna) was used to translate the entire cDNA from a compiled sequence of HTLV-1-PR:Fc construct in pDR2ΔEF1α.hFc vector into the corresponding amino acid sequence (XbaI, HTLV-I PR, NotI, Linker, Fcγ1)

Fig. 6

Immunofluorescence staining of CHO cells that expressed HTLV-1-PR:hFcγ1 recombinant fusion protein. Red signal: CHO stained with ethidium bromide and green signal: HTLV-1-PR:hFcγ1 recombinant fusion stained with FITC antibody

Fig. 7

A Fluorescence emission spectra. The substrate’s maximum relative fluorescent was 540 nm, which was 460 nm for the complex. B Increased fluorescence indicates proper enzyme activity in the vicinity of the substrate