In view of existing as well as potential future uranium pollution, the current complicated, time-consuming, inefficient and expensive treatment methods seem woefully out-of-date. To address these drawbacks, the Peking iGEM team put forward the Uranyl Reaper project, which aimed to develop an innovative method to deal with uranium pollution.
In order to achieve this goal, we designed an extraordinary biological adsorption polymer network which was produced by engineered bacteria and self-assembled completely autonomously. Based on the isopeptide bond formed between SpyCatcher and SpyTag, two designed crosslinking peptides, we developed triple SpyTag and SpyCatcher modules which could form a covalently-linked polymer network which offers high mechanical strength and high stability in diverse challenging environments.
This polymer network further gains the capacity to adsorb uranium in aqueous environments with high efficiency via the addition of a super uranyl binding protein (SUP) module. The SUP could also be replaced with other functional elements such as additional metal binding proteins, or the monomerized streptavidin (mSA) module which could mediate the clearance of the self-assembled polymer network through biotin-avidin interactions in order to achieve multi-functionality and modularization.
We utilized a series of signal peptides to ensure efficient secretion. A schematic graph of the whole project is shown in Fig. 1.
Local Concentration Enhancement - Crosslinking
To unite at least two functions in a single protein polymer network - uranium adsorption and recovery - we needed a protein polymer network which contains several functional modules. Inspired by the autocatalytic formation of the isopeptide bond between a Lys and an Asp residue in the CnaB2 protein from S. pyogenes, researchers engineered a pair of complementary truncations of CnaB2, called SpyTag and SpyCatcher1. These engineered peptides were able to form isopeptide bonds with each other spontaneously. In order to use the SpyTag-SpyCatcher system as scaffold, we fused three SpyTag and three SpyCatcher modules, respectively. the results indicated that the crosslinking reaction between the Triple SpyCatcher and fusion proteins could be completed in 1 hour.
Reaping the ions - Uranyl Adsorption
Considering that uranium mainly exists as uranyl ion and its carbonate in aqueous solution, we applied the Super Uranyl Binding Protein (SUP), which was designed specifically to bind uranyl via structural calculation and functional modification. SUP is thermodynamically stable and has a very high affinity and selectivity for uranyl ions, with a Kd of 7.4 femtomolar (fM) and >10,000-fold selectivity over other metal ions2. In our experiments, SUP retained its high uranyl affinity after being fused with the triple SpyTag protein, as well as in the covalently cross-linked protein polymer network. Impressively, in a trial where the concentration of uranyl was set at 10μM, the polymer network with the SUP module was able to sequester nearly 90% of total uranyl ions within just one minute.
Retrieving the network - Clearance
How to collect the protein polymer network together with the adsorbed uranyl from the environment? We focused on monomerized streptavidin (mSA), which binds biotin with a very low Kd value of less than 1nM3, and added it as a module to the polymer network in the form of a triple SpyTag-mSA fusion protein.
We next ligated biotin molecules to amino-coated magnetic beads, and when we used a magnet to immobilize these beads, the protein polymer network full of bound uranyl ions could be retrieved with an efficiency of up to 70%.
Simplification of protein purification - Secretion
To reduce costs and simplify the time-consuming procedures necessary to purify proteins from bacteria, we added secretion modules. Whether in E. coli or B. subtilis, secretory proteins usually contain signal peptides that are essential for their export from the cytoplasm. Which signal peptide would best promote the secretion of our constructs was inscrutable, so a library was built in order to screen for the most suitable candidates. We developed two assays (Western Blot and FIAsH fluorescence) to detect successful secretion. Finally, signal sequences derived from OmpA, LTIIb and PhoA were found to be efficient mediators for the secretion of our recombinant proteins. The fusion proteins could be brought to their working concentration by concentrating them directly in the culture supernatants, and the resulting crude concentrates could be used directly for the ultimate goal - the Uranium Reaper.
References:
[1] Zakeri, B., et al., Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences, 2012. 109(12): p. E690-E697.
[2] Lu, Z. et al. A protein engineered to bind uranyl selectively and with femtomolar affinity. Nature chemistry 1856, 236-241 (2014).
[3] Sau-Ching, W. et al. Engineering monomeric streptavidin and its ligands with infinite affinity in binding but reversibility in interaction. Proteins 77, 404-412 (2009).
