Multiple roles of the cellulase CBHI in enhancing production of fusion antibodies by the filamentous fungus Trichoderma reesei

Abstract

The production of Fab antibody fragments in Trichoderma reesei can be increased over 50-fold by fusing the core-linker region of the T. reesei cellulase CBHI (cellobiohydrolase I) to the heavy Fd chain (Nyyssönen et al. 1993). This beneficial role of CBHI in antibody production has now been studied further by comparisons of T. reesei trains producing the light chain only. Fab or CBHI-Fab all of which exhibited identical light chain integration. The N-terminal fusion of CBHI to the heavy Fd chain not only aided secretion, as expected, but also increased the level of mRNA encoding the CBHI-heavy Fd chain, either by stabilizing the messenger or by enhancing transcription. The CBHI part appeared to facilitate secretion at least by aiding the passage through the endoplasmic reticulum, since processing of the signal peptide of the antibody chains seemed to be most efficient in the strain producing CBHI-Fab in contrast to the strains producing light chain or Fab fragment. Interestingly, CBHI core-linker protein, originating from the CBHI-heavy Fd chain, was found in large amounts in the culture medium. The cleavage resulting in this tailless CBHI occurred inside the cell. This suggests that, by omitting the heterologous tail, the secretion of the resulting CBHI core-linker protein is enhanced to a level comparable with secretion of the extracellular T. reesei proteins.

High-Yield Production of a Bacterial Xylanase in the Filamentous Fungus Trichoderma reesei Requires a Carrier Polypeptide with an Intact Domain Structure

“…Fusions to both well-expressed full-length proteins and their catalytically active N-terminal domains (lacking the binding domains) have been successful. Positive effects of such gene fusions on the production of heterologous proteins have been observed at the (post)translational level and the transcriptional level (13,21,31). In T. reesei, fusions to the core-hinge domain of the major cellobiohydrolase I, Cel7A, have increased the production levels of calf chymosin (J. Uusitalo, unpublished data; referred to in reference 32), interleukin-6 (32; J. Demolder, X. Saelens, M. Penttilä, W. Fiers, and R. Contreras, Abstr.…”

Antibody production with yeasts and filamentous fungi: on the road to large scale?

“…In T. reesei pdiA transcript levels were increased 2-3 fold due to Fab over-expression (Nyyssonen and Keranen 1995). Over-expression of scFvLYS (directed against lysozyme) and scFv4715 (raised against Streptococcus sanguis; Frenken et al 1998) leads to a 5-to 10-fold increase in relative bipA mRNA levels compared to a wild type in A. awamori.…”

“…More than a 50-fold increase of Fab production could be achieved in T. reesei by fusing the heavy Fd chain (V H +C H 1 domains) to T. reesei cellulose CBHI (cellobiohydrolase I; Nyyssonen et al 1993). When examining the beneficial role of the fusion strategy, Nyyssonen and Keranen (1995) found that the N-terminal fungal protein does not only facilitate secretion by easing the passage through the ER but also lead to an increased level of mRNA coding for CBHIheavy Fd chain (by stabilizing mRNA or enhancing transcription), leading to titers up to 150 mg l -1 in a bioreactor. The gene fusion strategy also enhanced the secretion of some single chain fragments in A. awamori, where A. niger glycoamylase served as a carrier protein (Frenken et al 1998).…”

Approaches for refining heterologous protein production in filamentous fungi

“…The increase varies from 5 to 1000-fold, depending on the protein, resulting in protein levels varying from 5 to 250 mg l -1 Even higher levels, up to 1-2 g l -1 were obtained for bovine chymosin (Dunn-Coleman et al 1991) and lactoferrin (Ward et al 1995) when high-level-production strains were subjected to several rounds of mutagenesis. The production of antibody fragments in T. reesei as a CBH1 fusion, resulted in more than 150-fold increase in the yield (Nyssonen and Keranen 1995).…”