title: A method for in vitro synthesis of unglycosylated recombinant complement component C9.
abstract: A method for in vitro synthesis of human complement component C9 has been established in order to generate unglycosylated normal and mutant proteins without the need to sub-clone. One or two step polymerase chain reaction (PCR) was used to add the T7 RNA polymerase promoter and introduce multiple mutations within the cDNA. The cDNA was then transcribed by T7 RNA polymerase and the mRNA translated in a rabbit reticulocyte lysate or wheat germ system. Successful synthesis was confirmed by: the correct size of PCR product DNA on agarose gel electrophoresis, incorporation of [alpha-32P]UTP into mRNA, and formation of [35S]methionine-labelled protein of the correct molecular mass for full length C9. The wheat germ extract generated up to 1.5 micrograms of recombinant C9. This unglycosylated C9 had at least 10% of the haemolytic activity of native C9. Unglycosylated C9 polymerised more readily than the native protein. This spontaneous polymerisation was increased by removal of the first 23 amino acids or mutating two cysteines at positions 33 and 36. This therefore provides a rapid method for screening the effect of multiple mutations on the biological activity and polymerisation of pore forming proteins.
authors.affiliation: Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, UK.
authors.forename: K M
authors.initials: KM
authors.lastname: Taylor
authors.forename: J P
authors.initials: JP
authors.lastname: Luzio
authors.forename: A K
authors.initials: AK
authors.lastname: Campbell
mesh_terms: D000818:Animals; D001483:Base Sequence; D010856:Columbidae; D003186:Complement C9 / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D003165:Complement System Proteins / Q000276:immunology; D018076:DNA, Complementary / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D006031:Glycosylation; D006461:Hemolysis; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D014764:Viral Proteins
chemical_list: D003186:Complement C9; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C042295:complement C5b-8 complex; D003165:Complement System Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases