Part:BBa_K5398601

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Name: Mussels foot protein type 6 (Mfp6)

Base Pairs: 363 bp

Origin: Escherichia coli, synthetic

Properties: A protein for maintaining the reducing conditions needed for optimal wet adhesion in marine mussels

2. Usage and Biology

Mussels foot protein type 6 (Mfp6), which has a molecular weight of 13.8 kDa, is a protein found in the byssal gland cells of mussels. It belongs to the family of mussel foot proteins and plays a crucial role in maintaining the reducing conditions necessary for optimal wet adhesion in marine mussels. Mfp6 is particularly rich in cysteine, a sulfur-containing amino acid that can form stable structures in mussel adhesion proteins and provide antioxidant protection. This antioxidant property helps prevent over-oxidation of dopamine (DOPA) residues in adhesion proteins, thereby maintaining their adhesive function.

During the adhesion process of mussels, Mfp6 may work in conjunction with other foot proteins, such as Mfp3 and Mfp5, which are rich in dopamine and are key factors in the adhesive strength of mussel adhesion proteins. Mfp6, through its antioxidant properties, helps protect dopamine residues at the initial stage of adhesion, thus maintaining the protein's adhesive ability. In addition, Mfp6 may also regulate the tautomeric balance of adhesion proteins, further affecting adhesion performance.

Fig. 1 Mechanism of action.

Strains and Plasmid Construction

Plasmid Construction

The mfp6 sequence(363 bp) was cloned from the pETDuet-1-Mfp6 vector using a combined gradient and touchdown polymerase chain reaction(PCR) method. Specific primers were synthesized for PCR amplification(Table 1). The forward primer was designated as Mfp6-top, and the reverse primer as Mfp6-bottom. These primers, along with 2×Phanta Max Master Mix (Dye Plus), were used in a touchdown PCR reaction for 30 cycles with a temperature profile of 15 seconds at 95°C, 15 seconds at 56°C, and 1 minute at 72°C.

Similarly, the pET28a(+) sequence(5725 bp) was cloned from the pET28a(+)-TRn4-Mfp5 vector. The forward primer was pET28a(+)-top and the reverse primer was pET28a(+)-bottom(Table 1). The touchdown PCR reaction was performed for 30 cycles with a temperature profile of 95°C for 15 seconds, 67°C for 15 seconds, and 72°C for 1 minute.

Table 1. Sequence of Primer

The amplification products were analyzed by electrophoresis on 1% agarose gels stained with ethidium bromide(Fig. 2). An approximately 300 bp-specific Mfp PCR product was inserted into a pCR pET28a(+) vector for sequencing using the ClonExpress Ⅱ One Step Cloning Kit.

Fig. 2 1 % agarose gel electrophoresis of the PCR amplified Mfp6 and pET28a(+) vector. Line 1:5000bp DNA Marker. Lines 2-3: the PCR amplified Mfp6(363 bp). Lines 4-5: the PCR amplified pET28a(+) vector(5725 bp).

Transformation and Colony PCR

The final products named pET28a(+)-Mfp6 assembly underwent transformation into E.coli DH5a competent cells and then colony PCR was performed, using T7 and Mfp6 bottom primers(Table 1). For the colony PCR procedure, from the agar plate half amount of each colony was picked and diluted on 10 μl of doble distilled wate. 1 μL was used for sample preparation, while the remainder was used for liquid culture. The samples were loaded and run in 1% agarose gel electrophoresis and then we concluded that the recombination was successful(Fig. 3).

Fig. 3 a.The plasmid map of pET28a(+)-Mfp6. b.Colony PCR of E-coli DH5a transformants using T7 and Mfp6-bottom primers. Line: 2000 bp DNA Marker. Lines 2-9: pET28a(+)-Mfp6 using T7 and Mfp6-bottom primers (912 bp) from different colonies.

Sequencing

We selected colonies from Line 3 and Line 4 and performed overnight cultures in tubes containing 5 ml of medium. Subsequently, we extracted the plasmids using the FastPure Plasmid Mini kit and submitted them for sequencing. The sequencing results further confirmed the success of our recombination experiment(Fig. 4).

Fig. 4 Result of pET28a(+)-Mfp6 sequencing.

Cultivation, Purification and SDS-PAGE

Shaking Flask Cultivations

E. coli BL21(DE3) having the pET28a(+)-Mfp6 plasmid was grown in a shaking flask containing 50 mL of 2xTY medium and the culture conditions were set at 37℃ with shaking at 250 rpm. Cell growth was monitored by measuring the optical density at 600 nm (OD600) using a Nanodrop. When the OD600 reached 0.6 to 0.8, 10 μM IPTG (final concentration) was added to the culture to induce the expression of the recombinant Mfp6 protein. After induction, the cells were further cultivated at 37℃ for 5 hours before being centrifuged and lysed.

4.2 SDS-PAGE

From Fig. 5, we can know the Mfp6 protein with a molecular weight of 28 kDa was predominantly enriched in the pellet fraction, with the best results obtained when using Extraction buffer(5% v/v acetic acid, 50mM DTT, 8M urea) as the lysis buffer.

Fig. 5 SDS-PAGE of pET28a(+)-Mfp6(28 kDa). Line 1: Protein Marker. Line 2: Mfp6-Whole Cell Lysate(IPTG). Line 3: Mfp6-Supernatant(IPTG). Line 4: Mfp6-Pellet-PBS(IPTG). Line 5:Mfp6-Pellet-Extraction buffer(IPTG) . Line 6: Mfp6-Whole Cell Lysate-1. Line 7: Mfp6-Supernatant-1. Line 8: Mfp6-Pellet-PBS-1. Line 9:Mfp6-Pellet-Extraction buffer-1. Line 10: Mfp6-Whole Cell Lysate-2. Line 11: Mfp6-Supernatant-2. Line 12: Mfp6-Pellet-PBS-2. Line 13:Mfp6-Pellet-Extraction buffer-2.

Shaking Flask Cultivations

To obtain a larger quantity of protein, we cultured the target strain using the same method as described above, but with a 500 mL volume of 2xTY medium.

Purification and SDS-PAGE

Mfp6 was extracted from the pellet with Extraction buffer (5% v/vacetic acid, 50mM TCEP, 8M urea) and the supernatant was dialyzed overnight against 5%v/v acetic acid in a total volume ratio of 1:1200. Then, we used a HisTrap FF Crude column for immobilized metal affinity chromatography (IMAC) purification of the samples. First, the column was equilibrated with 5 resin volumes of washing bufferand then loaded with 5 ml of the resuspended denatured samples. Target recombinant Mfp6 was eluted with Elution buffer (50 mM Na2HPO4, 8 M Urea, 100 mM NaCl, 250mM/500mM Imidazole, pH 7.4)(Fig. 6). Eluted Mfp6 was dialyzed in 5 % v/v acetic acid overnight at 4°C, stored at -20℃.

Fig. 6 SDS-PAGE of pET28a(+)-Mfp6(28 kDa). Line 1: Protein Marker. Line2: Extraction buffer. Line 3: Supernatant. Line 4: Elution buffer (50 mM Na2HPO4, 8 M Urea, 100 mM NaCl, 50mM Imidazole, pH 7.4). Line 5: Elution buffer (50 mM Na2HPO4, 8 M Urea, 100 mM NaCl, 100 mM Imidazole, pH 7.4). Line 6: Elution buffer (50 mM Na2HPO4, 8 M Urea, 100 mM NaCl, 250 mM Imidazole, pH 7.4). Line 7: Elution buffer (50 mM Na2HPO4, 8 M Urea, 100 mM NaCl, 500 mM Imidazole, pH 7.4).

Activity Analysis of Mfp6

Activity analysis at different substrate concentrations

We conducted the reaction with varying concentrations of tyrosine as the substrate, adding an equal and sufficient amount of tyrosinase TyrVs to each well. The mixture was incubated at 37°C to allow for a full 30-minute reaction. Subsequently, an equal and excess amount of Mfp6 was added, and the reaction was allowed to proceed for an additional 5 minutes. The absorbance at 475 nm was then measured using a plate reader. As depicted in Figure 7, the OD values of the experimental group were consistently lower than those of the control group across all concentrations. This suggests that Mfp6 indeed reduced some of the dopaquinone back to dopamine, with the reduction effect being more pronounced at higher substrate concentrations.

Fig. 7 Result of Activity analysis. a.Mfp6 Activity analysis at different substrate concentrations. b.Mfp6 Activity Analysis on a 96-Well Plate.

Activity analysis at different reaction time

We utilized 750 μM tyrosine as the substrate in the reaction, adding an equal and appropriate amount of tyrosinase TyrVs to each well. The reaction was allowed to proceed at 37°C for a full 30 minutes. Following this, an equal and excess amount of Mfp6 was introduced, and the absorbance at 475nm was measured at 30-second intervals using a plate reader. As shown in Fig. 8, the OD values of the experimental group gradually decreased over time, while those of the control group remained virtually unchanged. This indicates that Mfp6 progressively reduced dopaquinone back to dopamine as the reaction progressed. Additionally, the difference in OD values between the experimental and control groups at the zero-minute mark may be attributed to unavoidable errors such as variations in sample addition time.

Fig. 8 Mfp6 Activity analysis at different reaction time.

6.Reference

[1] Nicklisch SC, Das S, Martinez Rodriguez NR, et al. Antioxidant efficacy and adhesion rescue by a recombinant mussel foot protein-6[J]. Biotechnol Prog, 2013, 29(6):1587-1593.