title: Optimisation of expression and purification of the feline and primate foamy virus transmembrane envelope proteins using a 96 deep well screen.
abstract: The production of recombinant transmembrane proteins is due to their biochemical properties often troublesome and time consuming. Here the prokaryotic expression and purification of the transmembrane envelope proteins of the feline and primate foamy viruses using a screening assay for optimisation of expression in 96 deep well plates is described. Testing simultaneously various bacterial strains, media, temperatures, inducer concentrations and different transformants, conditions for an about twentyfold increased production were quickly determined. These small scale test conditions could be easily scaled up, allowing purification of milligram amounts of recombinant protein. Proteins with a purity of about 95% were produced using a new purification protocol, they were characterised by gel filtration and circular dichroism and successfully applied in immunological assays screening for foamy virus infection and in immunisation studies. Compared to the previously described protocol (M. Mühle, A. Bleiholder, S. Kolb, J. Hübner, M. Löchelt, J. Denner, Immunological properties of the transmembrane envelope protein of the feline foamy virus and its use for serological screening, Virology 412 (2011) 333-340), proteins with similar characteristics but about thirtyfold increased yields were obtained. The screening and production method presented here can also be applied for the production of transmembrane envelope proteins of other retroviruses, including HIV-1.
authors.affiliation: Robert Koch Institute, Nordufer 20, Berlin 13353, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Mühle
authors.affiliation: German Institute for Cancer Research, Im Neuenheimer Feld 242, Heidelberg 69120, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Löchelt
authors.affiliation: Robert Koch Institute, Nordufer 20, Berlin 13353, Germany. Electronic address: DennerJ@rki.de.
authors.forename: Joachim
authors.initials: J
authors.lastname: Denner
mesh_terms: D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D057166:High-Throughput Screening Assays; D010957:Plasmids; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012521:Sarcosine / Q000031:analogs & derivatives; D012995:Solubility; D016092:Spumavirus / Q000235:genetics*; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C025231:sarkosyl; D005982:Glutathione Transferase; D012521:Sarcosine