We had already solved the self-assembly and uranyl-binding problems. However, the question how to retrieve the biological functional hydrogel from the environment remained unsolved.
We found that the reaction between streptavidin and biotin might be a good option. Biotin could be attached to amino-coated magnetic with relative ease. However, streptavidin could form a tetramer and bind to four biotin molecules with a Kd value of about 10−14M. The conjugation product of streptavidin and biotin is resistant to extreme pH values, denaturing agents, and enzymatic degradation1.
Although the streptavidin-biotin system is quite robust, the peculiarity of very tight tetramerization might restrict the use of native streptavidin and it might be difficult for four biotin molecules to get close because of the steric hindrance from other monomers in the hydrogel. We thus looked for monomeric streptavidin analogues.
A monomeric streptavidin (mSA) was constructed and characterized by another group in previous work going back to 2013. The Kd value and thus the biotin-binding affinity of mSA is less than 1nM, which is the highest affinity reported among monomeric streptavidins. The monomer also has a significantly higher stability and solubility than any of previously engineered monomers, which is necessary in order to ensure that the molecule remains folded and functional during its application in harsh environmental pollution scenarios. Each of the four independent mSA molecules could bind with biotin one-to-one, which is ideal for the use in our hydrogel (Fig. 1.)2.
In order to make the hydrogel retrievable, a recombinant protein consisting of mSA and 3A was designed. This way the mSA module could participate in the formation of the hydrogel as a triple SpyTag-mSA.
We ligated biotin molecules to amino-coated magnetic beads and when we used a magnet to immobilize these beads, the protein hydrogel full of uranyl ions could be efficiently retrieved. A schematic of the entire protocol is shown in Fig. 2.
- Protocols for Biotin-coated Magnetic beads
- Measure the binding efficiency of mSA with biotin-coated beads
- Measure the binding efficiency of mSA with Atto488 Biotin and Ultra Centrifugal Filters
- Measure the binding efficiency of mSA with Atto488 Biotin and Affinity chromatography
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It was important for us to know if the fusion protein, triple SpyTag-mSA, functioned as envisioned. We mixed 4μL of a suspension of either biotin-coated magnetic beads (10mg/ml) or recycled beads which were boiled and washed 3 times with PBST containing 1M NaCl, with a 40μL aliquot of a 1mg/ml solution of the triple SpyTag or triple SpyTag- mSA fusion protein, respectively. The reaction mixtures were incubated at 37°C for 1 hour under constant shaking at 1500 rpm. Vibration was necessary to avoid aggregation of the beads. After incubation, we took 10μL samples for SDS-PAGE analysis and a Bradford protein concentration assay, respectively. These two assays were used to determine the clearance efficiency of the mSA module.
In Fig. 3. (A), the color depth of lane 2 and lane 3 was remarkable lighter than lane 1. There were no differences between lanes 4, 5, and 6. This indicates that 3A-mSA could be efficiently bound to the biotin coated beads but 3A could not, as expected. The Bradford assay was used to quantitatively measure the concentrations of residual proteins in the solutions. Only 14% of 3A-mSA remained after contacting with biotin-coated magnetic beads for an hour. If the beads were boiled and washed, the binding efficiency was reduced and 27% of 3A-mSA remained unbound (data not shown). On the other hand, if the protein did not contain an mSA module, such as in the case of the triple SpyTag, the binding efficiency would be quite low, and 78.4% of 3A remained unbound after contacting with fresh beads.
These qualitative and quantitative results demonstrated that the protocols we used were functional, and that the biotin-coated beads could exhibit an excellent binding capacity and combine with mSA modules at a high efficiency, as well as with high specificity. It has also been shown that the beads remain usable after boiling and washing, with only a 15% loss of adsorption capacity. What's more, it could be shown that the biotin-coated beads could be recycled in order to reduce costs and achieve an overall more environmentally friendly process.
We demonstrated that the modules used to construct the 3A-mSA fusion protein remained functional. However, we still did not know if the polymerized hydrogel containing the mSA modules could be cleared. We thus mixed three kinds of monomers together and waited for 1 hour to let them crosslink as far as possible. To evaluate the biotin binding efficiency of the Spy Crosslinking Network, the resulting hydrogel was incubated with biotin-coated magnetic beads at 37°C for 1 hour under constant agitation at 1500 rpm in a shaker. The corresponding sample list is shown in Fig. 4.
The Bradford assay was used to measure the concentration of remaining unbound protein and the results showed that unbound protein remaining after contacting with the beads was much less than in the controls (Fig.5. A). Indeed, a marked decrease of protein concentration was visible when incubating the reaction system with beads for 1h, with only 22.1% of protein remaining unbound (Fig.5. B). This meant that the biotin-coated beads could be combined with the mSA module while retaining their functionality, even when incorporated into the protein hydrogel.
To assess if other protein modules in the hydrogel could be linked via the mSA module, we did another experiment to test the change of fluorescence intensity of an mRFP module incubated with or without beads. The experimental procedures were the same as above. The excitation wavelength used for mRFP was 584 nm, and the emission peak was 607 nm. The fluorescence intensity was measured on a plate reader, and the results are shown in Fig. 6.
The fluorescence intensity of control groups corresponded to about 12.8 AU while in the experimental groups this value was 3.19 AU. Only 24.9% of total fluorescence remained unbound, indicating that a majority of 3A-RFP modules could be assembled into the hydrogel, and that the 3A-RFP module could be retrieved via binding of the hydrogel via the 3A-mSA module. These results indicated that if we use 3A-SUP in this hydrogel, the biotin-coated beads could also be used to retrieve it, together with the bound uranyl cations.
We found that monomeric streptavidin, or mSA, which is able to tightly bind biotin, is a great candidate to retrieve the Spy Crosslinking Network and thus Uranium Reaper from the environment. The results demonstrated that the designs were effective. Not only could the monomeric triple SpyTag- mSA be recovered, but the hydrogel which contained mSA modules showed the same behavior, with a retrieval efficiency of about 80%. In further implementations using the 3A-mSA, we could thus exchange the 3A-mRFP, as illustrated in fig.4. , for a 3A- module, which could adsorb uranyl, in order to sequester uranium from the environment. In this way the special biological functional hydrogel would solve the pollution problem.
References:
[1] Christopher, D. et al. Streptavidin–biotin technology: improvements and innovations in chemical and biological applications. Appl Microbiol Biotechnol 97, 9343–9353 (2013).
[2] DeMonte D. et al. Structure-based engineering of streptavidin monomer with a reduced biotin dissociation rate: Streptavidin Monomer with a Reduced Off Rate. Proteins: Structure, Function, and Bioinformatics, 81(9), 1621-33 (2013).
