Part:BBa_K5398605:Experience

Applications of BBa_K5398605

We use the plasmid pET-28a(+)-Mfp6 to express the Mfp6 protein(BBa_K5398601) which can reduce the excessively oxidized dopaquinone back to Levodopa(L-DOPA), thereby enhancing the adhesive performance of the fusion protein TRn4-Mfp5(BBa_K5398020).

Characterization

Strains and Plasmid Construction

Plasmid Construction

The Mfp6 sequence(363 bp) was cloned from the pETDuet-1-Mfp6 vector using a 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 PCR reaction for 30 cycles with a temperature profile of 15 sec at 95°C, 15 sec at 56°C, and 1 min at 72°C.

Similarly, the pET-28a(+) sequence(5725 bp) was cloned from the pET-28a(+)-TRn4-Mfp5 vector. The forward primer was pET-28a(+)-top and the reverse primer was pET-28a(+)-bottom(Table 1). Polymerase Chain Reaction(PCR) was performed for 30 cycles with a temperature profile of 95°C for 15 sec, 67°C for 15 sec, and 72°C for 1 min.

Table 1 | Details of Plasmids、Fragments and Primers.

The purified fragments were analyzed by electrophoresis on 1% agarose gels stained with with YeaRed Nucleic Acid Gel Stain(Fig. 2). The Mfp6 fragment was inserted into the pET-28a(+) vector fragments using in-fusion cloning.

Fig. 2 | 1 % agarose gel electrophoresis of the PCR amplified Mfp6 and pET-28a(+) vector.

Line 1: 5000 bp DNA Marker; Lines 2,3: the PCR amplified Mfp6(363 bp); Lines 4,5: the PCR amplified pET-28a(+) vector(5725 bp).

Transformation and Colony PCR

The final plasmid named pET-28a(+)-Mfp6 assembly underwent transformation into E.coli DH5a competent cells and then colony PCR was performed, using T7 universal primer and Mfp6 bottom primers(Tab. 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 | The results of transformation and colony PCR.

a.The plasmid map of pET-28a(+)-Mfp6. b.Colony PCR of E-coli DH5a transformants using T7 universal primer and Mfp6-bottom primer. Line 1: 2000 bp DNA Marker; Lines 2-9: pET-28a(+)-Mfp6 using T7 top 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 LB medium. Subsequently, we extracted the plasmids using the FastPure Plasmid Mini kit and submitted them for sequencing. The sequencing result shows there was a synonymous mutation at the SUMO-tag site which did not influence the structure and function of Mfp6, while other sequences appeared normal.(Fig. 4).

Fig. 4 | Result of pET-28a(+)-Mfp6 sequencing.

Cultivation, Purification and SDS-PAGE

Shaking Flask Cultivations

E. coli BL21(DE3) having the pET-28a(+)-Mfp6 plasmid was grown in a shaking flask containing 50 mL of LB 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 h before being centrifuged and lysed.

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, 50 mM DTT, 8 M urea) as the lysis buffer.

Fig. 5 | Expression of pET-28a(+)-Mfp6(28 kDa).

a.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. b.Western blot of pET-28a(+)-Mfp6(28 kDa). Line 1: Mfp6-Whole Cell Lysate(IPTG); Line 2: Mfp6-Supernatant(IPTG); Line 3: Mfp6-Pellet-PBS(IPTG).

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 LB medium.

Purification and SDS-PAGE

Mfp6 was extracted from the pellet with Extraction Buffer (5% v/v acetic acid, 50 mM DTT, 8 M 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 Hypur T Ni-NTA 6FF (His-Tag) Prepacked Chromatographic Column, 1mL for immobilized metal affinity chromatography (IMAC) purification of the samples. First, the column was equilibrated with 5 resin volumes of washing buffer and 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, 250 mM 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 pET-28a(+)-Mfp6(28 kDa).

Line 1: Protein Marker; Line 2: Extraction Buffer; Line 3: Supernatant; Line 4: Elution Buffer(50 mM Imidazole); Line 5: Elution Buffer(100 mM Imidazole); Line 6: Elution Buffer(250 mM Imidazole); Line 7: Elution Buffer(500 mM Imidazole).

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-min reaction. Subsequently, an equal and excess amount of Mfp6 was added, and the reaction was allowed to proceed for an additional 5 min. The absorbance at 475 nm was then measured using a microplate reader. As depicted in Fig. 7, the OD values of the experimental group were consistently lower than those of the control group across all concentrations. Besides, an increasingly obvious colour occurs in experimental group compared to control group along with the increase of concentration. This suggests that Mfp6 indeed reduced some of the dopaquinone back to L-DOPA, 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 min. Following this, an equal and excess amount of Mfp6 was introduced, and the absorbance at 475 nm was measured at 30-sec intervals using a microplate 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 L-DOPA as the reaction progressed.

Fig. 8 | Mfp6 Activity analysis at different reaction time.

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.

[2] TAN D, ZHAO J P, RAN G Q, et al. Highly efficient biocatalytic synthesis of L-DOPA using in situ immobilized Verrucomicrobium spinosum tyrosinase on polyhydroxyalkanoate nano-granules [J]. Appl. Microbiol. Biotechnol., 2019, 103(14): 5663-78.

[3] YAO L, WANG X, XUE R, et al. Comparative analysis of mussel foot protein 3B co-expressed with tyrosinases provides a potential adhesive biomaterial [J]. Int. J. Biol. Macromol., 2022, 195: 229-36.

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