Part:BBa_K3089023
T7 promoter+csgA-linker-mfp5-mfp5-His
The csga-linker-mfp5-mfp5-his fusion protein is the purified version of Csga-linker-Mfp5 recombinant protein by His-tag affinity purification (a -Histidine-Histidine-Histidine-Histidine-Histidine-Histidine-Histidine tag fused on the carboxyl-terminal). We designed this part by putting together two identical Mfp5 mussel proteins and hoped it shows higher adhesive features than Csga-Mfp5. CsgA is an amyloid-like protein encoded on genome of E.coli MG1655 providing cohesive mechanical strength, and Mfp5 is a mussel foot protein from Mytilus galloprovincialis responsible for interface adhesion. This recombinant protein would self-assemble into fibrous bundles or films with adhesive properties by displaying the mussel adhesion domains on the surface of amyloid scaffolds, which would be a promising new generation of bio-inspired adhesives for a wide range of applications. This part was based on the paper "Strong underwater adhesives made by self-assembling multi-protein nanofibres" (Zhong et al., 2014) and has been characterised by iGEM15_TU_Delft (BBa_K1583104).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 47
Illegal PstI site found at 400 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 400
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 585
Illegal BamHI site found at 813 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 47
Illegal PstI site found at 400 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 47
Illegal PstI site found at 400 - 1000COMPATIBLE WITH RFC[1000]
Characterization
This recombinant protein is our own new design which has a property of both cohesion (CsgA) and adhesion (Mfp5). By adding two Mfp5s to CsgA, we expect it to have a stronger adhesive property. The results show, this recombinant protein can adhere to plastics and glasses better than any other parts in our toolbox. Therefore, this design cannot only help with the design of underwater adhesives but can also be used to make underwater living material. We characterised this part using various qualitative and quantitative data from protein expression, purification, and functional analysis.
- protein expression
- protein purification
- Surface coating analysis
Protein expression
csgA-linker-mfp5-mfp5 was cloned into pET28b and expressed in E.coli BL21(DE3) Rosetta by 500μM IPTG for 5h at 37℃. In order to detect its expression, whole cells were collected after induction by centrifuging and prepared for SDS-PAGE. Results showed that no obvious protein bands of CsgA-mfp5-mfp5(~25 kDa) could be observed on lane WC compared with lane pET28b (pET28b empty vector), which means the expression of this protein is not well in BL21(DE3) Rosetta.
Protein purification
For we make producing underwater bio-adhesives as the final goal of our project, though no obvious protein bonds of interest could be observed, we straightly went on protein purification.
CsgA is an amyloid-like protein characterised by β-strands, and CsgA monomers would form aggregates after expression inside cells. Therefore, denature protein purification methods were used.
Weak bands presented on the lane E2. The mixed solutions were then loaded on the columns and dialysed with PBS buffer (PH=6.0) to wash away imidazole. Meanwhile, the protein was concentrated.
After that, the concentrated protein was put under 4℃ for 72 hours to make it renature, protein concentrations of CsgA-linker-mfp5-mfp5 were measured by BCA assay, and its yield is 0.5mg/L.
Surface coating analysis
After obtaining a small number of recombinant proteins, surface coating analysis for qualitatively assessing the surface adsorption ability of recombinant proteins was conducted on two of most commonly used bio-related surfaces: hydrophilic glass slides and hydrophobic polystyrene tissue culture plates. As shown in Figure3, rBalcp19k-linker-mfp5 recombinant protein showed higher surface absorption abilities on both different substrates than rBalcp19k without fusion of mfp5 on its C-terminal. It’s suggested that Mfp improves the coating ability of rBalcp19k-linker-mfp5 fusion proteins. The In-vitro DOPA modification by mTyr-CNK tyrosinase significantly improved its surface absorption abilities, which suggested the positive contribution of DOPA in adhesive protein performances.
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