Production in yeast of pseudotype virus-like particles harboring functionally active antibody fragments neutralizing the cytolytic activity of vaginolysin

Abstract

BackgroundRecombinant antibodies can be produced in different formats and different expression systems. Single chain variable fragments (scFvs) represent an attractive alternative to full-length antibodies and they can be easily produced in bacteria or yeast. However, the scFvs exhibit monovalent antigen-binding properties and short serum half-lives. The stability and avidity of the scFvs can be improved by their multimerization or fusion with IgG Fc domain. The aim of the current study was to investigate the possibilities to produce in yeast high-affinity scFv-Fc proteins neutralizing the cytolytic activity of vaginolysin (VLY), the main virulence factor of Gardnerella vaginalis.ResultsThe scFv protein derived from hybridoma cell line producing high-affinity neutralizing antibodies against VLY was fused with human IgG1 Fc domain. Four different variants of anti-VLY scFv-Fc fusion proteins were constructed and produced in yeast Saccharomyces cerevisiae. The non-tagged scFv-Fc and hexahistidine-tagged scFv-Fc proteins were found predominantly as insoluble aggregates and therefore were not suitable for further purification and activity testing. The addition of yeast α-factor signal sequence did not support secretion of anti-VLY scFv-Fc but increased the amount of its intracellular soluble form. However, the purified protein showed a weak VLY-neutralizing capability. In contrast, the fusion of anti-VLY scFv-Fc molecules with hamster polyomavirus-derived VP2 protein and its co-expression with VP1 protein resulted in an effective production of pseudotype virus-like particles (VLPs) that exhibited strong VLY-binding activity. Recombinant scFv-Fc molecules displayed on the surface of VLPs neutralized VLY-mediated lysis of human erythrocytes and HeLa cells with high potency comparable to that of full-length antibody.ConclusionsRecombinant scFv-Fc proteins were expressed in yeast with low efficiency. New approach to display the scFv-Fc molecules on the surface of pseudotype VLPs was successful and allowed generation of multivalent scFv-Fc proteins with high VLY-neutralizing potency. Our study demonstrated for the first time that large recombinant antibody molecule fused with hamster polyomavirus VP2 protein and co-expressed with VP1 protein in the form of pseudotype VLPs was properly folded and exhibited strong antigen-binding activity. The current study broadens the potential of recombinant VLPs as a highly efficient carrier for functionally active complex proteins.

Production of recombinant VP1-derived virus-like particles from novel human polyomaviruses in yeast

“…The vector pFX7, without any insert or constructed plasmids, pFX7-KIPyV-VP1, pFX7-WUPyV-VP1, pFX7-MCPyV-VP1, pFX7-HPyV6-VP1, pFX7-HPyV7-VP1, pFX7-TSPyV-VP1, pFX7-HPyV9-VP1, pFX7-HPyV10-VP1, pFX7-STPyV-VP1, pFX7-HPyV12-VP1n 380 , pFX7-HPyV12-VP1s 380 , pFX7-HPyV12-VP1n 364 , pFX7-HPyV12-VP1s 364 , and pFX7-NJPyV-VP1 were transformed into the S. cerevisiae strain, AH22-214 ( a, leu2-3,112, his4-519 ). The growing conditions for yeast transformants harboring plasmids with VP1-encoding genes from different HPyVs and conditions for induction of VP1 synthesis were similar to that previously described [ 75 ]. Briefly, yeast cells were first cultured in glucose- then galactose-containing induction media for 24 and 18 h, respectively.…”

“…Recombinant VP1 protein purification was carried out as previously described [ 75 ]. Briefly, yeast cells containing recombinant VP1 protein were suspended in DB450 buffer (450 mM NaCl, 1 mM CaCl 2 , 0.001 % 0.25 M L-Arginine, and Trition X-100, in 10 mM Tris/HCl-buffer, pH 7.2) with 2 mM PMSF and EDTA-free Complete Protease Inhibitor Cocktail tablets (Roche Diagnostics, Mannheim, Germany), and then were homogenized with glass beads using Bead-Beater GB26 (BioSpec Products, Inc., Bartlesville, USA).…”

“…The membranes were blocked with 5 % milk in TTBS (0.1 % Tween 20, 0.1 M Tris, 0.3 M NaCl, pH 7.4) and incubated overnight in the relevant antibody solutions at room temperature. Anti-JCPyV, −MCPyV, and -WUPyV VP1 mouse sera (raised against the respective VP1 protein) and anti-HaPyV VP1 rabbit serum [ 75 ] were diluted 1:500, 1:300, 1:300, and 1:1000, respectively, in TTBS. After incubation with the diluted antibodies, the membranes were incubated for 2 h with horseradish peroxidase-labeled anti-mouse or anti-rabbit IgG conjugate (Bio-Rad, Hercules, CA, USA) diluted 1:1000.…”

Recombinant antibody production evolves into multiple options aimed at yielding reagents suitable for application-specific needs

“…7 ). Such structures represent multivalent antibody nanoparticles possessing strong antigen-binding and -neutralizing activities that effectively prevented mammalian cell lysis induced by vaginolysin [ 56 ]. Another approach considers the Saccaromyces display of scFvs fused to non-self-cleaving Mxe GyrA intein for promoting the chemical functionalization of the antibody fragment by exploiting the Expressed Protein Ligation methodology.…”

Construction of polyomavirus-derived pseudotype virus-like particles displaying a functionally active neutralizing antibody against hepatitis B virus surface antigen

“…Hamster polyomavirus (HaPyV) VP1 protein has been exploited for the generation of either chimeric VLPs harbouring foreign epitopes, or pseudotype VLPs when co-expressed with the minor capsid protein VP2 that binds within the central 5-fold cavity of each VP1 pentamer [ 8 – 11 ]. Moreover, it was demonstrated that HaPyV-derived pseudotype VLPs represent an efficient carrier for functionally active complex molecules, such as antibodies [ 12 ]. In these pseudotype VLPs, an intact VP1 protein is functioning as a carrier mediating VLP formation of both VP1 and the modified VP2 protein molecule.…”