Tryptophan-tagged cutinase studied by steady state fluorescence for understanding of tag interactions in aqueous two-phase systems

Genetic engineering has been used to construct fusion proteins of Fusarium solani pisi cutinase and tryptophan-based tags, expressed in Saccharomyces cerevisiae, to increase the partitioning in aqueous two-phase systems. The separation systems were composed of thermoseparating polymers (random copolymers of ethylene oxide and propylene oxide, EOPO) and detergents (C(12)EO(n)). In this study, the fluorescence behaviour of the peptide-tagged protein, free peptide tag and tryptophan was investigated. The tryptophan-tagged proteins, cutinase-(WP)(4) and cutinase-TGGSGG-(WP)(4), showed emission spectra similar to the free peptides and tryptophan, indicating solvent exposure of the tag. The influence of polymers and detergents on the fluorescence of tagged proteins was examined. When peptides and tagged proteins were exposed to polymer, a slight blue shift of the emission maximum was observed. Larger blue shifts of the emission maximum were observed when C(12)EO(n) detergents were utilised. The results correlate with aqueous two-phase partitioning where addition of C(12)EO(n) detergents results in more extreme partitioning compared to systems containing only polymers. Dynamic light scattering (DLS) measurements of the EOPO copolymers were carried out, showing that the polymers did not aggregate at concentrations used in aqueous two-phase systems. Quenching of fluorescence with iodide for both proteins and peptide tags was studied. Plots according to the Stern-Volmer equation resulted in a linear fit, indicating exposed tryptophan residues for both free peptides and fusion proteins. The quenching constants were similar for both tagged protein and free peptide tag. The fluorescence results indicated that the tryptophan residues in the tag were exposed to the solvent and could interact with detergents and polymers in the two-phase systems.