Print Email Facebook Twitter Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (VHH) R9 coupled to Arthromyces ramosus peroxidase (ARP) Title Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (VHH) R9 coupled to Arthromyces ramosus peroxidase (ARP) Author Joosten, V. Roelofs, M.S. van den Dries, N. Goosen, T. Verrips, C.T. van den Hondel, C.A.M.J.J. Lokman, B.C. TNO Kwaliteit van Leven Publication year 2005 Abstract The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5′- or 3′-terminal ends of the gene encoding llama variable heavy chain antibody fragment VHH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which produced up to 30 mg l-1 fusion protein in the culture medium. Both fusion proteins showed peroxidase activity in an ABTS activity test. Considerable amounts of fusion protein were detected intracellularly, suggesting that the fungus encounters problems in secreting these kind of proteins. ELISA experiments showed that ARP-R9 was less able to bind its antigen, the azo-dye RR6, as compared to R9-ARP. Furthermore, in contrast to R9-ARP, ARP-R9 bound to RR6 did not show peroxidase activity anymore. These results indicate that fusion of ARP to the C-terminus of the antibody fragment VHH R9 (R9-ARP) is the preferred orientation. © 2005 Elsevier B.V. All rights reserved. Chemicals / CAS: peroxidase, 9003-99-0; Antibodies, Monoclonal; Fungal Proteins; Immunoglobulin Heavy Chains; Immunoglobulin Variable Region; Peroxidase, EC 126.96.36.199; Recombinant Fusion Proteins Subject BiologyBiotechnologyAntibodiesAntigensCytologyDyesEnzyme kineticsEnzymesFungiGenesProteinsMagic BulletsArthromyces ramosus peroxidase (ARP)Aspergillus awaromiFusion proteinsHeterologus protein productionLlama variable heavy chain antibody fragments (VHH)Genetic engineeringhybrid proteincarboxy terminal sequencecomparative studyculture mediumenzyme activityenzyme linked immunosorbent assaygene cassettegenetic codegeneticsnonhuman protein bindingprotein expressionAnimalsAntibodies, MonoclonalAspergillusCamelids, New WorldFungal ProteinsFungi, UnclassifiedImmunoglobulin Heavy ChainsImmunoglobulin Variable RegionPeroxidaseRecombinant Fusion ProteinsArthromyces ramosusLama glama To reference this document use: http://resolver.tudelft.nl/uuid:adb43ffc-a7e6-49c9-8dc4-65e385e11350 DOI https://doi.org/10.1016/j.jbiotec.2005.06.034 TNO identifier 239016 ISSN 0168-1656 Source Journal of Biotechnology, 120 (4), 347-359 Document type article Files To receive the publication files, please send an e-mail request to TNO Library.