1. Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (VHH) R9 coupled to Arthromyces ramosus peroxidase (ARP)
- Author
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B. Christien Lokman, Niels van den Dries, Vivi Joosten, Theo Goosen, C. Theo Verrips, Marc S. Roelofs, Cees A. M. J. J. van den Hondel, and TNO Kwaliteit van Leven
- Subjects
fusion ,domain ,Immunoglobulin Variable Region ,gene cassette ,Biochemistry ,Applied Microbiology and Biotechnology ,Lama glama ,thaumatin production ,genetics ,Dyes ,comparative study ,Fungal protein ,biology ,filamentous fungi ,Antibodies, Monoclonal ,heterologous expression ,General Medicine ,Enzymes ,enzyme activity ,genetic code ,light-chains ,secretion ,Aspergillus ,Aspergillus awaromi ,saccharomyces-cerevisiae ,Immunoglobulin Heavy Chains ,Camelids, New World ,Biotechnology ,Peroxidase ,culture medium ,nonhuman protein binding ,Heterologus protein production ,Recombinant Fusion Proteins ,Magic Bullets ,Biochemie ,Bioengineering ,Immunoglobulin light chain ,Antibodies ,Fungal Proteins ,Fungi, Unclassified ,Arthromyces ramosus peroxidase (ARP) ,Animals ,Antigens ,gene ,Biology ,protein expression ,Aspergillus awamori ,hybrid protein ,carboxy terminal sequence ,Heavy-chain antibody ,Enzyme kinetics ,Fungi ,Proteins ,endoplasmic-reticulum ,Llama variable heavy chain antibody fragments (VHH) ,Molecular biology ,Fusion protein ,enzyme linked immunosorbent assay ,Fusion proteins ,Genes ,Genetic engineering ,biology.protein ,Immunoglobulin heavy chain ,Arthromyces ramosus ,Heterologous expression ,Cytology - 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 1.11.1.7; Recombinant Fusion Proteins
- Published
- 2005
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