Difference between revisions of "Part:BBa K4247021"

Revision as of 12:12, 3 October 2022

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.

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.

@@ Line 39: / Line 39: @@
 '''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 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.
+'''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.
 [[File: sdst.jpeg|700px|]]