Expression of UC and CU chimaeras in E. coli
The UC and CU chimaeras were successfully constructed and transformed into E. coli BL21. To determine the optimum expression conditions, the effect of temperature and inducer concentration were investigated. The best result was obtained with 1 mM IPTG at 20 °C and overnight incubation.
The protein content of induced and non-induced recombinant strains was analysed using SDS-PAGE. Accordingly, the production of recombinant protein under the control of the T7 promoter led to the appearance of a sharp band in the soluble fraction of the cell lysate (Fig. 1). Although a molecular mass of 72 kDa was expected for both chimaeras, the expressed proteins exhibited a mobility corresponding to a molecular mass between 35 and 40 kDa. The analyses of the diluted sample revealed two distinct bands for uricase (~ 34 kDa) and intein–caleosin (~ 40 kDa). Subsequently, the activity of the produced proteins was confirmed by uricase assay. Apparently, cleavage of the fusion protein has occurred under the reducing conditions used for SDS-PAGE. The faster migration of intein–caleosin could be caused by the binding of caleosin to available Ca2+ (Chen et al. 1999). In contrast, the multi-subunit structure of uricase is supposed to show less mobility on SDS-PAGE since they are resistant to denaturation by SDS (Pitts et al. 1974).
It is noteworthy that membrane proteins like caleosins are insoluble and get stuck in the pellet. However, the chimaera proteins were soluble.
Purification of uricase by AOB system
The purification of recombinant uricase was conducted using the AOB-based system. In the first step, AOBs were successfully formed using oil, phosphatidylcholine, and soluble fraction of cell lysate (with and without the chimaera proteins). The construction of AOBs was confirmed by microscopic visualization. As can be seen in Fig. 2, the AOBs were almost coalescence in the control condition, which was without the chimaera proteins. However, the presence of UC protein makes the nanoparticles (about 0.2 μm) (Fig. 2C). The comparison between the chimaera proteins indicates that CU is not a competent candidate for the construction of AOBs (Fig. 2B). It seems a free C-terminus might be required for caleosin to build more stable and smaller droplets. However, it has been reported that the N-terminus is also necessary for targeting caleosin to oil bodies (Purkrtová et al. 2015).
After centrifugation, the AOBs separated from the aqueous phase and washed to eliminate the non-specific proteins (Fig. 3, lanes 2 and 3). Then, uricase was released by inducing the self-cleaving intein through shifting the pH and/or using 40 mM DTT at room temperature. Accordingly, uricase was retrieved in the aqueous phase (Fig. 3, lane 4), whereas intein–caleosin (Fig. 3, lane 1) remained in AOBs. Finally, all the mentioned fractions were resolved on SDS-PAGE (Fig. 3). Repetition of purification steps improved the purity of the uricase (Fig. 3, lane 5).
The specific activity of the enzyme was increased from 0.53 U/mg in the crude extract to 15.2 U/mg in the purified enzyme, which is comparable with the purified uricases by chromatography columns (Fazel et al. 2014; Khaleghi and Asad 2021).
AOB stability
The AOB particles are shaped by balancing the attractive and repulsive forces of structural proteins (Tzen et al. 1992). Therefore, any change in pH, ions or solvent that causes protein unfolding affects emulsion stability.
Our investigations on a wide range of pH values (3 to 11) revealed phase separation at the pH around the isoelectric point (pI) of caleosin (pH 5). Indeed, a gradual increase in the emulsifying property occurs as the pH value gets far from the isoelectric point (Wang et al. 2019; Gao et al. 2021). Moreover, the AOB droplets aggregate at pH 3 to 4 since the acidic environment increases surface hydrophobicity of oil bodies and thus leads to coalescence (Gao et al. 2021).
The effects of three concentrations of 50, 100, and 300 mM of different salts, including MgCl2, CaCl2, KCl, and Na2SO4, were also traced on AOB suspension for 15 min by turbidity tests. All the salts caused instability proportional to the ionic strength (Fig. 4A). However, the rapid reduction in turbidity by 0.3 M CaCl2 (p < 0.05) could occur as a result of the interaction of calcium with the EF_hand motif placed on the N-terminal domain of caleosin. This Ca2+-binding motif responds to biotic and abiotic stresses and plays a role in releasing triacylglycerols from oil bodies during seed germination (Poxleitner et al. 2006; Partridge and Murphy 2009; Shimada and Hara-Nishimura 2010). Although calcium caused sedimentation of AOBs, its low concentrations (7.5 mM) have been used as a divalent to cross-link oil-body proteins and Pickering stabilizing (Liu et al. 2017). It has been reported that the Pickering emulsions need an oil volume fraction (φ) of greater than 0.2 (Guo et al. 2021).
In contrast to salts, surfactants are generally considered to act as emulsifying agents because of reducing surface tension, breaking hydrophobic interactions of proteins and increasing elasticity, viscosity, and electronegative repulsion (Sukhotu et al. 2014). Although using 2% Tween 20 had no positive effect on AOB stability, adding 0.1% SDS rendered the suspension more stable (Fig. 4B).
A comparison between the stability of AOBs at 4 °C and room temperature showed that the lower temperatures prevent the emulsion coalescence (Fig. 4B), even if adding 0.1 M salts (Fig. 4C). However, sensitivity to divalent cations (Ca2+, Mg2+, and Zn2+) is not affected by temperature changes (Fig. 4C). The same results have been reported for emulsions containing calcium or magnesium (Ramkumar et al. 2000; Romero-Guzmán et al. 2020). Our further investigations indicated that AOBs without the chimaera protein also show the same instability towards divalent salts (data not shown). Therefore, the reason for emulsion instability differs depending on the ionic strength and oil volume fraction.
Effect of C- and N-terminal fusion on uricase activity
Two chimaeras, CU and UC, were designed to study the effect of C- and N-terminal fusion on uricase activity. As shown in Fig. 5, the enzyme activity was lower in UC chimaeras (p < 0.05).
To evaluate the binding affinity of the chimaera proteins to the uric acid, the 3D structure was modelled using Phyre2. Subsequently, 88% and 92% of residues of CU and UC were represented at > 90% confidence (Fig. 6). Furthermore, a docking study by default parameters of SwissDock revealed no binding at the expected points of UC chimaera. However, several binding sites were predicted for CU chimaera, and one of them included the expected residues (ΔG = − 6.97 kcal/mol). Indeed, uricase consists of 301 residues in which the catalytic triad (T57* K10* H256) were delimited by the six conserved residues (R176-Q228, N254-T57, and F159) (Gabison et al. 2008). As it is shown in Fig. 6, most of the defined residues (except T57* K10*) were identified on CU chimaera.