title: Calcium influences the stability and conformation of rotavirus SA11 glycoprotein VP7 expressed in Dictyostelium discoideum.
abstract: We have previously reported expression of the rotavirus outer capsid glycoprotein, VP7, in the relatively new expression host, Dictyostelium discoideum. To optimise yields of recombinant VP7, we examined the role of Ca2+ since stability of both VP7 and mature rotavirus during a rotavirus infection are calcium-dependent. Low micromolar levels of free extracellular Ca2+ were required to maximise yields of VP7 in D. discoideum whilst levels of VP7 were reduced following depletion of intracellular Ca2+ reserves using A23187 and EGTA. Immunoblot analysis suggested that VP7 was being degraded in an intracellular compartment. Immunoprecipitation with a conformation-dependent neutralising antibody confirmed that EGTA-induced Ca2+ chelation alters the conformation of VP7. These results suggest that stability of VP7 is dependent on maintaining adequate levels of intracellular Ca2+ and that conformational changes in VP7 which occur following depletion of Ca2+ reserves induce rapid proteolysis of the protein. Since these results establish conditions for expressing optimal levels of VP7 in the correct conformation they have important implications for the development of a subunit vaccine based on recombinant VP7.
authors.affiliation: MUCAB (Macquarie University Centre for Analytical Biotechnology), School of Biological sciences, Macquarie University, Sydney, N.S.W., Australia.
authors.forename: K R
authors.initials: KR
authors.lastname: Emslie
authors.forename: M B
authors.initials: MB
authors.lastname: Coukell
authors.forename: D
authors.initials: D
authors.lastname: Birch
authors.forename: K L
authors.initials: KL
authors.lastname: Williams
mesh_terms: D000818:Animals; D000956:Antigens, Viral; D001709:Biotechnology; D002021:Buffers; D002118:Calcium / Q000494:pharmacology*; D002213:Capsid / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D036022:Capsid Proteins; D003470:Culture Media; D004023:Dictyostelium / Q000235:genetics; D004355:Drug Stability; D006801:Humans; D010710:Phosphates; D011487:Protein Conformation / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012401:Rotavirus / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D014614:Vaccines, Synthetic / Q000302:isolation & purification; D014765:Viral Vaccines / Q000302:isolation & purification
chemical_list: D000956:Antigens, Viral; D002021:Buffers; D036022:Capsid Proteins; D003470:Culture Media; D010710:Phosphates; D011994:Recombinant Proteins; C050779:VP7 protein, Rotavirus; D014614:Vaccines, Synthetic; D014765:Viral Vaccines; D002118:Calcium