3.1. Use of PhaP as a fusion partner for immobilization of enzymes
Due to its high affinity for PHB (Zhao et al., 2016) and relatively small size compared to phasins from other bacterial species (Table S3), we investigated the use of PhaP from Aeromonas hydrophila as an affinity tag to immobilize proteins on PHB surfaces. In the initial experiments, we examined whether fusion with PhaP affected the expression of target proteins. When PhaP-sfGFP and sfGFP-PhaP were expressed in E. coli strain BL21(DE3), both fusion proteins were produced at similar levels to those of the parental sfGFP (Figure 1A, upper panel). Similarly, expression of LipM7 lipase was not significantly affected by N- or C-terminal fusion with phasin (Figure 1B, upper panel), indicating that PhaP could be fused upstream or downstream of target proteins without affecting the efficiency of gene expression.
The location of the PhaP fusion could significantly affect the biological activity of target proteins. In the case of sfGFP, the fusion protein with C-terminal PhaP displayed ~33% higher fluorescence than the control sfGFP (Figure 1A, lower panel). It has been reported that the functionality of a fluorescence protein can be enhanced when fused with a foreign protein sequence (Solovyov et al., 2011, Huang et al., 2016). However, the enzymatic activity of LipM7 exhibited greater dependence on the location of phasin fusion. Although LipM7-PhaP was not significantly different from wild-type LipM7 in terms of its ability to hydrolyze pNPD, PhaP-LipM7 exhibited <25% the activity of LipM7 (Figure 1B, lower panel). Although the structure of this lipase has not been determined, this result suggests that the N-terminus of this enzyme plays an important role in catalytic activity, in accordance with previous results showing that lipase activity can be disrupted when fused to N-terminal fusion partners (Gustavsson et al., 2001; Singh et al., 2018; Malunavicius et al., 2018).