Part:BBa_K4247021

Usage and Biology

Mussels have the ability to attach themselves to various surfaces underwater by permanent adhesion. This adhesion is facilitated by their byssus, which is secreted from their foot. The byssus comprises a bundle of threads and at the end of each thread, there is an adhesion plaque containing a water-resistant adhesive that enables the mussel to anchor itself to surfaces.

Several types of foot proteins have been characterised and each of them have a different function as per their location in the byssus. Of these, MFP3 and MFP5 are found in the distal end of the byssus. Post-translational modification of tyrosines yields L-3,4-dihydroxyphenylalanine (DOPA) and these DOPA groups are associated with the adhesion strength of MFPs. MFP3 and MFP5 are known to have the highest DOPA content among MFPs and hence, these intrinsically disordered proteins enable the adhesion mechanisms of the byssus. MFP1 forms the outer coating of the byssus and it has a lower DOPA content compared to MFP3 and MFP5.

Recombinant production of MFP5 wasn’t very successful and there were several bottlenecks in terms of cell growth and protein purification since the sticky nature of the protein makes it difficult to purify. In order to overcome these limitations, a hybrid protein called MFP151 was constructed and produced. This hybrid protein consists of six M. galloprovincialis MFP1 decapeptide repeats added to the N- and C-terminus of M. galloprovincialis MFP5. So, the protein consists of 6 MFP1 repeats followed by a MFP5 sequence and 6 MFP1 repeats again. MFP151 was found to have comparable adhesion characteristics to recombinant MFP5, could be produced with greater yields and could be purified easily.

Characterization

Addition of SnoopCatcher to Mfp151

Aim - The tyrosine residues in the Mfp151 protein undergo post-translational modifications (PTMs) to become 3,4-dihydroxyphenyl-alanine (Dopa) which provides the Mfp151 proteins with their adhesion properties. Since E.coli is not capable of performing PTMs, this can be overcome by co-expressing another plasmid producing tyrosinase to modify tyrosine residues to Dopa in vivo. Mfp151 and Mfp151_Snoopcatcher were expressed. Further, they were also co-expressed with tyrosinase to facilitate the post-translational modification of tyrosine to Dopa to make the proteins adhesive.

Results - In the SDS-gel, it is difficult to visualise the Mfp151 protein. So, a western blot was done on the above SDS-gel.

Conclusion - Thus, we can see the expression of Mfp151 (25kDa), Mfp151_Snoopcatcher (37kDa) along with tyrosinase (30kDa) in the co-expression cultures. We cannot see orf438 (cofactor) because it ran out of the gel. In the next experiment however it's visible.

Protein purification by IMAC

Aim - To purify the protein by IMAC (immobilized metal ion chromatography) using Ni-NTA resin.

Results - Mini columns were loaded with Ni-NTA resin and the soluble fraction of the lysate was added to the columns. Then, the columns were washed twice and eluted to obtain the purified protein. An SDS-gel was run with the different purification fractions and a western blot was done on the gel. It is clear that most of the protein was lost in the flowthrough and washes and nothing was eluted, showing that the proteins did not bind to the Ni-NTA column at all.

Lysis buffer - 10mM Tris-Cl, 100mM NaH2PO4, 8M Urea, pH 8

Wash buffer - 10mM Tris-Cl, 100mM NaH2PO4, 8M Urea, pH 6

Elution buffer 1 - 10mM Tris-Cl, 100mM NaH2PO4, 8M Urea, pH 4.5

Dialysis buffer - distilled water

Conclusion - Although a considerable amount of the protein was lost in the flowthrough and washes, it is clear that the protein is eluted with high purity.

Co-transformation with tyrosinase and cofactor

Aim - To show that the co-transformation of the pET24 (+) vector containing the mfp151 (BBa_K4247018-BBa_K4247021) and the pRSET A vector containing tyrosinase (BBa_K4247023) and orf438 (tyr-cofactor) (BBa_K4247022) works.

Result - SDS and Western Blot was done on the purification fractions obtained from Ni-NTA purification of the protein from BL21(DE3) cells induced with 0.1 mM IPTG overnight. As it can be observed, in the SDS, tyrosinase (31.56 KDa) and orf438-cofactor- (16.48 KDa) are being produced. Since Mfp151 does not have any tryptophan residues, it is not possible to visualise Mfp151 proteins in an SDS-gel and hence, a western blot is needed.

So, a western blot was done on the above SDS-gel to confirm that the proteins we see are indeed the minispidroin proteins. Since the proteins were expressed with a 6x His-tag, we used mouse anti-hexa his primary antibodies and goat anti-mouse HRP-conjugated secondary antibodies for the western blot.

Conclusion - As seen in the Western Blot, we lost proteins in the flowthrough and washes. However, it still proves we managed to produce mfp151 and mfp151_SnoopCatcher in co-transformation with tyrosinase and orf438. The cofactor is clearly visible in the SDS-gel but not so clear in the Western Blot, possibly because the 6x HisTag is not well exposed.

NBT assay

Aim - To qualitatively evaluate the amount of tyrosine residues modified into DOPA-tyrosine and understand whether the co-expression system of mfp151 + orf438-tyrosinase operon made mfp151 more adhesive. The final goal was to test whether we could increase the interactions between mfp151 and minispidroins.

Results - We performed an NBT assay (Nitro-Blue Tetrazolium), where the purified and dialysed proteins (mfp151, mfp151 + tyrosinase coexpression, mfp151_SnoopCatcher and mfp151_SnoopCatcher + tyrosinase coexpression) were treated with a 0.6 mg/mL NBT solution and washed with a 0.16 M potassium glycinate solution.

Conclusion - Mfp151 is clearly the most stained protein, indicating that the co-expression system did not work effectively. This could be due to multiple reasons, such as a bias from other metal binding proteins that were not removed effectively during IMAC purification. After this result, we decided to implement a more precise protein conjugation tool as an alternative to making the Mfp151 more sticky.

Validation of SnoopCatcher's function

Aim - To demonstrate the spontaneous isopeptide bond formation between our minispidroin protein displaying the SnoopTag and Mfp151_SnoopCatcher.

Results - The proteins were allowed to interact with each other by mixing equal amounts of both proteins. The reaction was carried out at 25°C for 40 minutes and the pH of the reaction was maintained at 8 using pH adjusted PBS. After 40 minutes, the reaction was stopped by adding SDS sample buffer. Then, we performed an SDS-PAGE and a Western blot to visualize the results. We would expect to see a band whose molecular weight is the sum of the molecular weights of both proteins to indicate that both proteins are bound to each via an isopeptide bond between the SnoopTag and the SnoopCatcher. We see such a band only in the reaction where Mfp151_SnoopCatcher and Minispidroin 2rep with a SnoopTag were allowed to interact. The fact that we don't see this band in any of the controls confirms that the proteins were able to bind to each other solely due to the SnoopTag-Catcher system.

Conclsuion - The SnoopCatcher enabled the Mfp151_SnoopCatcher to bind to Minispidroin 2rep with a SnoopTag and hence, the function of the SnoopTag-Catcher system was validated.