title: Production in yeast of pseudotype virus-like particles harboring functionally active antibody fragments neutralizing the cytolytic activity of vaginolysin.
abstract: BACKGROUND
  Recombinant 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.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  Recombinant 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.
authors.affiliation: Vilnius University, Institute of Biotechnology, Department of Eukaryote Genetic Engineering, Graiciuno 8, LT-02241 Vilnius, Lithuania.
authors.forename: Milda
authors.initials: M
authors.lastname: Pleckaityte
authors.forename: Aurelija
authors.initials: A
authors.lastname: Zvirbliene
authors.forename: Indre
authors.initials: I
authors.lastname: Sezaite
authors.forename: Alma
authors.initials: A
authors.lastname: Gedvilaite
mesh_terms: D057134:Antibodies, Neutralizing / Q000235:genetics* / Q000276:immunology; D001426:Bacterial Proteins / Q000276:immunology*; D001427:Bacterial Toxins / Q000276:immunology*; D006194:Gardnerella vaginalis / Q000276:immunology; D015870:Gene Expression; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics* / Q000276:immunology; D009500:Neutralization Tests; D054353:Perforin / Q000235:genetics / Q000276:immunology*; D011120:Polyomavirus / Q000235:genetics; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology; D014764:Viral Proteins / Q000235:genetics* / Q000276:immunology
chemical_list: D057134:Antibodies, Neutralizing; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D007141:Immunoglobulin Fc Fragments; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D014764:Viral Proteins; C542392:vaginolysin, Gardnerella vaginalis; D054353:Perforin