Difference between revisions of "Part:BBa K4247019"

Revision as of 10:15, 10 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

Optimization of inducer concentration

Aim - To determine the concentration of inducer required for optimal protein expression.

Results - Cell cultures were grown ON at 37°C. Then, the next day, the cultures were diluted to an OD600 of 0.1 and induced with 0.1, 0.3, 0.5 and 1mM IPTG and grew ON. We would expect to see a band around 25kDa. However, this is a stain free SDS-gel that uses tryptophan residues to detect the protein. 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 - Thus, it is clear that 0.1mM IPTG is the optimal concentration for expression of Mfp151.

Optimization of lysis buffer

Aim - To determine the best buffer for cell lysis that provides most of the protein in a soluble state.

Results - In order to lyse our cells by sonication, we used 2 different lysis buffers to decide which lysis buffer gave the most proteins in the soluble fraction. The recipes of the buffers are as follows,

Buffer 1: 50 mM NaH2PO4 + 500 mM sodium chloride + 10 mM imidazole + 0.5% Triton X-100 + 10% glycerol + 2 mM DTT (added fresh, right before use), pH 8.0

Buffer 2: 10 mM Tris–Cl + 100 mM sodium phosphate, pH 8.0

The cell cultures were centrifuged to obtain the cell pellets which were resuspended in the cell lysis buffer and then sonicated until a clear lysate was obtained. The lysate was centrifuged to obtain the insoluble and soluble fractions in the pellet and supernatant respectively.

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

Conclusion - From the western blot, we can conclude that buffer 2 is better than buffer 1 since it provides most of the protein in the soluble fraction. This is further backed up by the results of the Mfp151 literature since the authors used buffer 2 as well.

Heat purification

Aim - To determine if there is a temperature that would precipitate only the Mfp151 proteins without the other E.coli proteins, to facilitate an easy purification method using heat treatment.

Results - The soluble fraction of the lysate was subject to different temperatures (40, 45, 70, 80°C). Then, after heat treatment, the samples were centrifuged and the supernatants and pellets obtained after different temperatures were run on an SDS-gel. In the SDS-gel, it is difficult to visualise the Mfp151 protein.

So, a western blot was done on the above SDS-gel. At 70 and 80°C, only a small amount of the Mfp151 protein precipitates in the pellet. Whereas at 40 and 45°C, a large fraction of the Mfp151 protein precipitated. However, it is not very pure as seen by the presence of other bands.

Conclusion - So, although most of the protein precipitates at temperatures at 40 and 45°C, heat treatment is not a suitable method for protein purification since there is no temperature that precipitates Mfp151 without also precipitating other proteins.

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

Since most of the protein was lost in the flowthrough and washes in the previous attempt, the soluble fraction containing the proteins was allowed to incubate with the Ni-NTA resin under shaking conditions for 30-60 mins. This would provide sufficient time for the resin to bind the protein. Further, due to the sticky nature of the protein, using harsh conditions like very low pH might be better at eluting the protein rather than using a high concentration of imidazole.

So, the columns were washed twice and eluted twice to obtain the purified protein wherein the second elution was done with 0.5M HCl. An SDS-gel was run on the different purification fractions and a western blot was done.

Conclusion - So, with a sufficient incubation time (30-60mins) that allows the Ni-NTA resin to bind all the proteins, no proteins are lost in the flowthrough and washes. Further, it is clear that harsh conditions are required for Mfp151 elution since most of the protein is eluted with 0.5M HCl.

Optimization of media

Aim - To determine which media, LB (Luria broth) or TB (Terrific broth) is better for protein expression.

Results - 5 different colonies (C1, C2, C3, C4, C5) of cells expressing the Mfp151 protein were grown in LB or TB in otherwise identical conditions. The cells were grown ON at 37°C and the cultures were induced with 0.1mM IPTG and allowed to express the protein ON.

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

Conclusion - It is clear that we get the most Mfp151 protein with C2 when it is grown in LB.

Protein yields

Aim - To do a BCA assay after dialysis of the protein to quantify the final yield.

Results -

Mfp151 was inoculated in LB, grown at 37°C, induced with 0.1mM IPTG and allowed to express the protein ON in a flask. Further, in order to produce large quantities of protein, the proteins were produced in large scale by cultivating the cells in a 1L fermenter. The cells were grown in 1L of batch media at 37°C to an OD600 of 77 which takes about 20h. Then, when the cells have consumed all the media, there is a spike in the oxygen saturation levels due to depletion of glucose. At this point, the cells were induced with 250uM IPTG and fed-batch (500ml) mode was started. Then, after 20h of induction, the cells were cultivated and the proteins were purified by IMAC.

Conclusion - The yield for Mfp151 was found to be 4.8mg/L of culture when grown in a flask. However, with fermentation, the yield increased to 5.61mg/L of culture.

Co-expression of tyrosinase

Aim - To co-express a functional tyrosinase in another plasmid along with the plasmid expressing the Mfp151.

Results - 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.

Conclusion - In the cells co-expressing Mfp151 and tyrosinase, we can clearly see a band at around 30kDa (expected size of tyrosinase). Thus, tyrosinase was co-expressed in vivo along with Mfp151. The cofactor (aorund 16 kDa) ran out of the gel, but it's visible in the next experiment.

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.