Production of bifunctional proteins by Aspergillus awamori: llama variable heavy chain antibody fragment (V(HH)) R9 coupled to Arthromyces ramosus peroxidase (ARP)

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 V_HH 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 V_HH R9 (R9-ARP) is the preferred orientation.