Team:Greatbay SCIE/Demonstrate

Demonstration

Sticky SticKit!

Microbial adhesive proteins assembly line

We constructed pET28b plasmid systems of CsgA-mfp5, CsgA-mfp5-mfp5, CsgA-mfp5-mfp3, Fp1-mfp5-fp1, rBalcp19K and rBalcp19K-mfp5. They were transformed into BL21(DE3) Rosetta for expression. They were grown to OD600 ~0.6 in LB broth containing 50 mg/mL kanamycin and 30 mg/mL chloramphenicol at 37°C. Protein expression was induced with 0.5 mM IPTG at 37°C for 5 h.

Figure 1. Coomassie-blue-stained SDS-PAGE analysis of Fp151 His-tag affinity purification under denaturing conditions. Lanes: M, protein molecular weight marker; NC, whole-cell sample of pET28b empty vector; WC, whole-cell sample of recombinant proteins; FT, flow through after resin binding; E, eluted proteins. (see details in results)

All protein were successfully expressed and purified while Fp151, rBalcp19k and rBalcp19k-Mfp5 had a relatively higher yield.

Table 2 Final yield of all recombinant adhesive proteins.

Functional characterization

DOPA (Dihydroxyphenylalanine) is a crucial unnatural amino acid that contributes to Mfp5/Mfp3 adhesion. Mfp5 was a core adhesion contributor protein in our toolbox. We have produced mTyr-CNK individually and tried in vitro DOPA modification. The protein yielded very high, approximately 7mg/L (3B), higher than any other recombinant protein in our toolbox. The mTyr-CNK in-vitro modification was assessed by NBT staining tests. All in-vitro modified recombinant proteins produced positive result (turned purple), suggesting that tyrosines of Mfp5 were successfully modified into DOPA, BSA protein was used as a negative control (Figure 3)

Figure 3 Purification of mTyr-CNK and test of in vitro tyrosine hydroxylation by NBT staining. (A) Cell pellets collected after protein expression. (B) SDS-PAGE of purified mTyr-CNK by affinity chromatography. NC: empty vector; mTyr-CNK: tyrosinase from marine microorganism; (C) NBT staining to detect tyrosine hydroxylation of recombinant protein Csg-mfp5, CsgA-mfp5-mfp5, Fp151 and rBalcp19k-mfp5(see details in results).

After obtaining a small number of recombinant proteins, using surface coating analysis (see methods), the qualitative assessment of the surface adsorption ability of recombinant proteins was performed on 2 of the most commonly used bio-related surfaces: hydrophilic glass slides and hydrophobic polystyrene tissue culture plates (Figure 4). Mfp5-related recombinant exhibited higher surface absorption abilities than other recombinant proteins, whereas almost all absorbed BSAs (negative controls) were washed away (Figure 5). DOPA modification significantly improved the surface absorption abilities of Mfp-related recombinant proteins (see details in results).

Figure 4 Surface coating analysis assay.
Figure 5 Surface coating analysis of recombinant proteins on hydrophilic glass slides (left) and hydrophobic polystyrene (PS) plates (right).

We tested the adhesion of recombinants proteins with plastics. As shown in Video(left), negative control BSA protein showed no adhesion to the plastic, while CsgA-Mfp5 (left 1) and CsgA-Mfp5-Mfp5 (left 4) was strongly adhesive, though Fp151(left 2) and rBalcp19k (left 3) were slightly less adhesive.

Video-Adhesion test between plastics.

Fp151 was further tested because it was easy to obtain. Results indicated that Fp151 proteins were more adhesive to plastic and glass while showing no obvious adhesion to rubber band fragments and pieces of paper.

Figure 6 Adhesion test of Fp151.

As we successfully obtained these recombinant proteins and conducted small scale characterization to understand their various properties, we are still eager to improve their yields to obtain more concentrated proteins in greater volumes. We hope our researches will inspire future iGEMers to utilize recombinant adhesive proteins in future projects.