Part:BBa_K1989000

1. Usage and Biology

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In the last few years, hydrogens made from natural or synthetic polymers have been investigated due to their extensive application in clinical medicine and synthetic biology. Comparing to traditional biological material, protein-based multifunctional biological material is low-cost, facile and eco-friendly. However, strategies for assembling 3D molecular networks synthesized only by protein molecular remain underdeveloped. The reason why investigating this technology is still tough is lack of protein-based cross linking agents. Inspiring from the self-catalysis of isopeptide bond between Lys and Asp in Streptococcus pyogenes fibronectin-binding protein FbaB, researchers split the catalytic domain and obtain two peptide called Spytag(the short one) and Spycatcher(the long one) which are able to form isopeptide bond with the other without any assistant. By fusing Spytag and Spycatcher with functional domains respectively, researchers solve the problem tactfully. In order to using Spytag and Spycatcher system as scaffold, we fused three Spytag spaced by (VPGVG)4 with 6xHistag in N-terminal and another functional protein called Super Uranyl-binding Protein(SUP) in C-terminal.

Another part of this CDS is uranyl-binding domain. Uranium is the key element for nuclear-energy production and is crucial in many other applications. The most stable and relevant uranium ion in aerobic environment is uranyl cation. Super Uranyl-binding Protein(SUP), a completely artificial protein from structure calculating to function modifying is designed to binding uranyl cation specifically. According to the researchers’ result, uranyl-binding affinity and selectivity of SUP is extremely high. The dissociation constant is lower than 10fM which is 1000 folds lower comparing to other common mental ions.

Based on our results, the fused protein His-3A-SUP(3A-SUP) possess both isopeptide bond forming function and uranyl-binding ability. Thus, using 3A-SUP as a part of hydrogel formation, we can obtain our multifunctional biomaterial.

2. Cultivation, Purification and SDS-PAGE 2.1 Cultivation The part was assembled with T7 promoter and RBS in pET28a plasmid vector. E. coli strain BL21(DE3) harboring the appropriate plasmid was grown at 37 °C in 2xYT medium overnight with suitable concentration of antibiotic. The culture was diluted 100 fold into fresh medium with antibiotic and grown at 37°C to an optical density of 0.6~0.8 at 600 nm, the protein expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and cells were grown overnight at 25°C.

2.2 Purification Cells were centrifuged at 8000rpm for 15min at 4°C. Resuspend the cell paste expressing recombinant protein in binding buffer (20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1mM β-mercaptoethanol, pH7.4), containing SIGMAFAST™ Protease Inhibitor Cocktail Tablets (SIGMA-ALORICH). Disrupt the cells with sonication for 20 min with suitable power on ice and centrifuge at 18000 rpm for 40 min at 4°C. Remove remaining particles by passing the supernatant through a 0.22 μm filter.

The HisTrap™ column (GE Healthcare, Inc.) was equilibrated with binding buffer. Load the sample and wash the column with binding buffer. Elute the target protein with a linear gradient starting with binding buffer and ending with the same buffer including 500mM imidazole. The eluted fraction containing the target protein were concentrated by Amicon® Ultra Centrifugal Filters (Merck) with a 10 kDa cutoff, then frozen by liquid nitrogen and stored at -80°C.

2.3 SDS-PAGE Protein purification was checked by SDS-PAGE and the resulting protein is quantified by Braford analysis.

3. Activity Analysis 3.1 Gel Formation 3.2 Uranyl-binding affinity and selectivity We tested the adsorption capacity of 3A-SUP,4A-SUP,6A-SUP and 3A-SUP+3B,4A-SUP+3B,6A-SUP+3B in TBS buffer(pH=7.02) against 10μM uranyl. We wanted to do some comparisons and found the best candidate. Cross-linked 3A-SUP+3B can sequester 95.77% of the total uranyl in TBS buffer (pH=7.0,10μM), showing the best adsorption capacity. Other proteins also can sequester at least 60% of the total uranyl. The standard deviation were calculated from triplicate experiments. We then test different protein-uranyl ratio in TBS buffer against 10μM uranyl. The use of one and ten equivalents of protein against one equivalent of uranyl (10μM) in TBS buffer can sequester 89.79% and 92.70% of the total uranyl respectively. When protein-uranyl ratio increased, the adsorption rate also increased, but not significantly. The standard deviation were calculated from triplicate experiments. To test whether our protein can work in real water condition, we use Weiming Lake and synthetic sea water as solutions. 3A-SUP+3B can sequester 89.79% and 26.88% and 89.93% of the total uranyl respectively in TBS buffer, Weiming lake water and synthetic sea water. The standard deviation were calculated from triplicate experiments. We find that when proteins were employed in lake water, the adsorption decreased, the reason might be that other creatures in lake water interferes the proteins’ function. Furthermore, we decreased the uranyl concentration to 13nM and increased the equivalent of the proteins to test the sensitivity of 3A-SUP+3B in synthetic TBS buffer and sea water. To test the adsorption sensitivity, thee use of 6000 equivalent of protein against one equivalent of uranyl (13nM) in synthetic sea water and TBS buffer can remove 33.11% and 47.48% respectively. The concentration was determined by ICP-MS.