title: Increased disulphide dimer formation of latent associated peptide fusions of TGF-β by addition of L-cystine.
abstract: The development of novel protein therapeutics relies on the ability to express appreciable amounts of correctly folded recombinant proteins. Latent IFN-β is engineered using the latency-associated peptide (LAP) of transforming growth factor β1 (TGF-β1) to maintain IFN-β in a biologically inactive form until such time as it is released at sites of inflammation by matrix metalloproteinase activity (see Adams et al., 2003). CHO cells cultured in suspension were used for expression of latent IFN-β to allow medium scale transient transfection. However, the recombinant protein expressed in this system consisted of a mixture of properly linked disulphide dimers and monomers. The ratio of dimer:monomer produced could be significantly altered towards increased dimer production by the addition of L-cystine to the CHO culture medium. The total yield of latent IFN-β was increased by co-transfection of plasmid coding for the simian virus (SV) 40 large T antigen to the plasmid with the SV40 origin of replication expressing latent IFN-β DNA. These results provide valuable new insights for developing protocols to produce substantial quantities of latent cytokine dimers in CHO cells in suspension.
authors: Bone and Joint Research Unit, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, United Kingdom.
authors: Lisa M
authors: LM
authors: Mullen
authors: Gill
authors: G
authors: Adams
authors: Yuti
authors: Y
authors: Chernajovsky
mesh_terms: D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D016466:CHO Cells; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006224:Cricetinae; D003470:Culture Media / Q000737:chemistry; D003553:Cystine / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D018628:Gene Dosage / Q000235:genetics; D016899:Interferon-beta / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism; D053779:Latent TGF-beta Binding Proteins / Q000378:metabolism*; D051379:Mice; D050505:Mutant Proteins / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D055503:Protein Multimerization / Q000187:drug effects*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D019163:Reducing Agents / Q000494:pharmacology; D013696:Temperature; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*
chemical_list: D000952:Antigens, Polyomavirus Transforming; D003470:Culture Media; D004220:Disulfides; D053779:Latent TGF-beta Binding Proteins; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D019163:Reducing Agents; D016212:Transforming Growth Factor beta; D003553:Cystine; D016899:Interferon-beta; D004247:DNA; D019704:Protein Disulfide-Isomerases