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

Appl Biochem Biotechnol. 2022 Nov 18;1-15. doi: 10.1007/s12010-022-04259-y. Online ahead of print.

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.