For all 4 constructs tested, calcium concentrations greater than 25 mM were found to cause precipitation of the fusion protein. To assess the ideal calcium concentration, all 4 constructs were precipitated from 1 mL of clarified cell lysate in 25, 50, 75, and 100 mM CaCl2. Pellets were washed in salt-free tris buffer five times. Pellets were broken up upon washing, but did not redissolve until exposed to an equivalent concentration of EGTA after the final wash. The 100 mM CaCl2 samples were found to not fully redissolve, so only lower CaCl2 concentrations were tested further. A slight increase in recovery was observed at 75 mM CaCl2 (as compared with lower CaCl2 concentrations) as confirmed by SDS-PAGE (data not shown). All 4 constructs were subsequently purified by precipitation with 75 mM CaCl2 and SDS-PAGE gels were run after 5 washes (Figure 4). No significant difference was found with increasing number of washes, so further quantification and recovery measurements were performed on samples washed five times. To confirm scalability, the analogous protocol was also performed on 50 mL lysate, and comparable results were obtained (data not shown). Additionally, we briefly tested the reversibility of the precipitation process. It was found that addition of calcium to the redissolved pellet in EGTA solution did yield a pellet once again. Full pellet size was only recovered after dialysis into EGTA-free buffer.
We next sought to quantify the recovery and functionality of the purified proteins after precipitation. To assess recovery of MBP-BRT17, we used the theoretically determined extinction coefficient to estimate concentration by absorbance at 280 nm (30). Results from purifying the construct on an amylose resin were compared with BRT precipitation. For MBP-BRT17-GFP,recoveries were calculated as the percentage of fluorescence signal of purified sample compared with lysate (this was normalized against control lysate). Along with total protein recoveries estimated by UV absorbance, recoveries of both MBP-BRT17-βlac and MBP-BRT17-AdhD were estimated by comparing lysate activity to the activity of these constructs after purification. MBP-BRT17-βlac recoveries were calculated using activity measured by tracking the absorbance at 486 nm for the hydrolysis of nitrocefin. MBP-BRT17-AdhD recoveries were calculated by tracking NADH formation at 340 nm in saturating conditions of both substrate and cofactor. Results of these trials are shown in Table 1. For MBP-BRT17, calcium precipitation recovers about double the amount of protein as compared with amylose resin purification. For MBP-BRT17-GFP, we observed up to 86% recovery of fluorescence. MBP-BRT17-βlac recovery from the lysate was not as high, but was still 5-fold better than the amylose resin, yielding a significant quantity of protein. Similar results were also observed for MBP-BRT17-AdhD, although the yields were not quite as high compared with the resin (2-fold improvement). It should be noted that while the overall values of the activities recovered in Table 1 appear low, they were all larger than the values obtained using the amylose resin purification. It is also possible that measuring activity in crude extracts may introduce error beyond what was accounted for in the measurement of endogenous hydrolysis (β-lactamase) and reduction (AdhD). Table 2 lists the absolute yield of each fusion protein based on UV absorption at 280 nm. All fusion proteins were shown to be purified in high yields.
To increase the utility of this tag, it would be beneficial to couple our system with a cleavage tag to separate the protein of interest from the BRT. The pMAL_c4E vector we used for these experiments contains a cleavable enterokinase site between the MBP and BRT. This recognition sequence was removed via site-directed mutagenesis. A new enterokinase site was engineered between the BRT and the protein of interest for MBP-BRT17-βlac and MBP-BRT17-AdhD. Therefore, as a proof of principle, we took precipitation purified MBP-BRT17-βlac and MBP-BRT17-AdhD and subjected them to overnight cleavage by enterokinase digestion. Calcium can then be added directly to the cleavage reaction to precipitate MBP-BRT17, thereby separating the tag from the protein of interest following centrifugation. This is shown in Figure 5 for MBP-BRT17-AdhD, showing pure, soluble protein by SDS-PAGE. Recoveries of 93 ± 7% were obtained by tracking UV absorbance at 280 nm, meaning 93% of the AdhD in the precipitation purified sample was recovered after cleavage and reprecipitation of the tag. Specific activity of the purified enzyme was also calculated to be 20.2 ± 1.3 min−1, which is similar to what has been previously reported, indicating this system has little to no effect on the activities of purified proteins (28). However, in the case of MBP-BRT17-βlac, the cleaved β-lactamase remained in the insoluble fraction following enterokinase cleavage and calcium precipitation. Upon further investigation it was found that β-lactamase will precipitate in high calcium concentrations. As a control experiment, we purchased recombinant β-lactamase and observed similar behavior. In 75mM CaCl2, an insoluble pellet was formed upon centrifugation. Activity assays confirmed a significant amount of active protein in the insoluble fraction (data not shown). This illustrates a caveat of the BRT system. Proteins that naturally precipitate in CaCl2 solutions cannot be efficiently separated from the BRT. For future improvement of this system, the protease used could be fused to the precipitating BRT or a self-cleaving intein could be incorporated. Fusing the protease to the BRT would enable its removal from the target protein in the final precipitation. A self-cleaving intein would fulfill a similar function. It should also be noted that the BRT can precipitate without being fused to the MBP, suggesting that the MBP is not essential for this system; however, the MBP may be useful for improving protein expression levels.