Expression and characterization of a tripartite fusion protein consisting of chimeric IgG-binding receptors and beta-galactosidase.
Using protein engineering, a tripartite fusion protein was constructed consisting of five IgG-binding regions of protein A from Staphylococcus aureus, two IgG-binding regions of protein G from Streptococcus strain G148 and beta-galactosidase from Escherichia coli. The resulting protein lacks the serum albumin binding regions of native protein G. The fusion protein, which is a tetramer of approximately 660 kDa, was designed as a tool for immunological assays taking advantage of its broad spectrum of antibody affinity. The gene was placed under control of two promoters, the PR promoter and the lac UV5 promoter and the expression from the two promoters was studied in a bioreactor. Induction of the PR promoter gave an intracellular product concentration corresponding to 20% of the cell dry weight. By utilizing the properties of beta-galactosidase, the protein was purified by extraction in an aqueous two-phase system. The fusion protein was not proteolytically degraded during the cultivation and purification steps. The biological activity of all three parts of the protein was demonstrated with a competitive ELISA.
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
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Strandberg
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Hober
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Uhlén
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Enfors
D000943:Antigens, Differentiation / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002678:Chimera / Q000235:genetics; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D005696:Galactosidases / Q000235:genetics*; D015870:Gene Expression; D007544:Isopropyl Thiogalactoside; D011401:Promoter Regions, Genetic; D011961:Receptors, Fc / Q000235:genetics*; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics*
D000943:Antigens, Differentiation; D001426:Bacterial Proteins; D004269:DNA, Bacterial; C034284:IgG Fc-binding protein, Streptococcus; D011961:Receptors, Fc; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D007544:Isopropyl Thiogalactoside; D005696:Galactosidases; D001616:beta-Galactosidase