Genetically engineered peptide fusions for improved protein partitioning in aqueous two-phase systems. Effect of fusion localization on endoglucanase I of Trichoderma reesei

Genetic engineering has been used for fusion of the peptide tag, Trp-Pro-Trp-Pro, on a protein to study the effect on partitioning in aqueous two-phase systems. As target protein for the fusions the cellulase, endoglucanase I (endo-1,4-beta-Dglucan-4-glucanohydrolase, EC 3.2.1.4, EGI, Cel7B) of Trichoderma reesei was used. For the first time a glycosylated two-domain enzyme has been utilized for addition of peptide tags to change partitioning in aqueous two-phase systems. The aim was to find an optimal fusion localization for EGI. The peptide was (1) attached to the C-terminus end of the cellulose binding domain (CBD), (2) inserted in the glycosylated linker region, (3) added after a truncated form of EGI lacking the CBD and a small part of the linker. The different constructs were expressed in the filamentous fungus T. reesei under the gpdA promoter from Aspergillus nidulans. The expression levels were between 60 and 100 mg/l. The partitioning behavior of the fusion proteins was studied in an aqueous two-phase model system composed of the thermoseparating ethylene oxide (EO)-propylene oxide (PO) random copolymer EO-PO (50:50) (EO50PO50) and dextran. The Trp-Pro-Trp-Pro tag was found to direct the fusion protein to the top EO50PO50 phase. The partition coefficient of a fusion protein can be predicted with an empirical correlation based on independent contributions from partitioning of unmodified protein and peptide tag in this model system. The fusion position at the end of the CBD, with the spacer Pro-Gly, was shown to be optimal with respect to partitioning and tag efficiency factor (TEF) was 0.87, where a fully exposed tag would have a TEF of 1.0. Hence, this position can further be utilized for fusion with longer tags. For the other constructs the TEF was only 0.43 and 0.10, for the tag fused to the truncated EGI and in the linker region of the full length EGI, respectively.