Part:BBa_K3755012

AKTK tag-his tag-2*Mfp5 CDS

Composite CDS to be put in protein expression plasmid(for example: pET28) to express 2*mfp5 protein in *E.coli* BL21

Usage and Biology

Mussels are magical creatures using their byssus to stick tightly to a variety of substrata that are wet, saline, corroded, and/or fouled by biofilms^[1]. We found that mussel foot protein (Mfp) is the key to the mussel's remarkable adhesion ability. The adhesion of mussel foot proteins is related to several molecular forces, among which the most important are the H-bond, π−π stacking interactions, and metal complexation between the 3,4-dihydroxyphenylalanine(dopa) group in the protein and the hydrophilic group on the material surface^[1,2,7].

Mfp is attractive not just for its great adhesion in water conditions, but for its superior biocompatibility as well^[3]. Up to now, Mfp has been well documented as an excellent wound healing dressing by many articles^[2,4,5]. Our project plans to add Mfp to the hydrogel to increase its mechanical strength and to link broken bone pieces. Since the product needs to be put into the internal environment, which takes high requirements for human safety, Mfp is exactly the best bioglue we can find. By comparison, in the Mfp family, Mfp-5 has the highest dopamine content(∼26 mol %) and the strongest adhesion^[1,6,7]. Therefore, we take the Mfp-5 as the best choice for our adhesion system.

In order to obtain high yield Mfp5 efficiently and conveniently, we chose to express recombinant Mfp5 in E.coli. Luckily, Dong Soo Hwang's team from Pohang University has isolated the Mfp5 sequence from an M. galloprovincialis foot cDNA library and successfully expressed the recombinant Mfp5 in E.coli BL21 in 2003^[8]. Later in 2018, Eugene Kim's team from Washington University in St. Louis referred to Dong Soo Hwang's research, using synthetic DNA with codons optimized for expression and making more amazing designs for Mfp5^[9]. The above results provide us a significant basis for us to produce Mfp5 with high yield and high viscosity.

The composite part is a complete 2 repeats of Mfp5 expression system, consisting of functional components and protein CDS. The AKTK expression tag((part:BBa_K3755009)) is at the start of the sequence to increase translation initiation rates, and the following His tag(part:BBa_K3755020) and linker(part:BBa_K3755021) plays roles in protein purification, and at the end of the sequence is the 2*Mfp5 CDS(part:BBa_K3755003). We add the start codon and the stop codon to the beginning and the end of the sequence, and put it into the plasmid pET28a. Because the promoter is T7 lac, after the plasmid is constructed, we transformed it into the E.coli BL21(DE3) strain and use IPTG to induce the protein expression.

Experiment and Results

Plasmid Construction

We got our expression plasmid pET28a-Mfp5-2 by placing an order with GENEWIZ.

Plasmid Transformation into BL21

E.coli BL21(DE3) competent strain was used for plasmid transformation. The method is as follows:

1) Add 1 µ l of pure plasmid solution to 100 µ l of competent bacteria solution.
2) Place the bacteria solution in ice and let stand for 30 minutes.
3) Thermal activation at 42 ° C for 90 seconds.
4) Add 600 µ l antibiotic free LB medium and incubate on a 37 ° C,220 rpm shaker for 45 minutes.
5) Centrifuge at 3500RPM for 5 min ,then remove 400 µ l supernatant.
6) Mix the remaining 200 µ l solution with the precipitated bacteria and then apply to 30µ l/ml kanamycin LB solid medium.
7) Culture at 37 ℃ for 12 hours.

Exploration of Cinditions of Induction

Before protein purification, we decided to explore the proper induction conditions. So we made two control groups and four experimental groups. In Figure 1, we could find that mfp5-1 was successfully expressed in four groups since there was an obvious band between 15-20 kDa compared with blank control(Blank BL21(DE3)) and negative control(Before induction).

Notably, there was a slight band in negative control(Before induction). We inferred that leaked expression of mfp5-1 might happen in negative control.

Fig.1: SDS-PAGE of mfp5-1 expression in different induction conditions

IPTG Inducion

After obtaining the single colony of transformed bacteria on the solid medium, we need to inoculate them in liquid culture to expand culture and then induce them with IPTG to get enough protein-producing bacteria:

1. the colony was inoculated into 5ml of 30µ l/ml kanamycin LB liquid medium and cultured on a 37 ° C,220 rpm shaker for 8 hours.
2. Then inoculated 5ml of bacterial solution in each 1L of 30µ L/mL kanamycin LB liquid medium.
3. When the OD value of the bacterial solution at 600nm reached 0.8-1.0, 500µ L IPTG solution of 1M was added to each L of bacterial solution, and the culture was performed on a 37 ° C,220 rpm shaker for 8-10 hours.
4. Finally, the obtained bacterial solution was centrifuged at 3500RCM for 20 minutes to obtain the precipitated bacteria.

Protein Purification

The protocol of Mfp5-2 purification is the same as Mfp5-1 purification since they share almost same features except molecular weight.

Since the recombinant Mfp5 expressed in E.coli is mostly in inclusion bodies, we need to add denaturants(here we used guanidine hydrochloride refer to the literature^[9]) during purification to maintain the soluble state of the protein. In order to obtain high purity protein, we added 6*His tag to Mfp5CDS and purified it by nickel ion affinity chromatography. The buffer formula and purification steps are as follows:

Buffer preparation

• Lysis buffer: 6 M guanidine hydrochloride, 50 mM potassium phosphates, 300 mM sodium chloride, pH 7.4
  
• Wash buffer: 6 M guanidine hydrochloride, 50 mM potassium phosphates, 300 mM sodium chloride, 50 mM imidazole, pH 7.4
  
• Elution buffer: 6 M guanidine hydrochloride, 50 mM potassium phosphates, 300 mM sodium chloride, 250 mM imidazole, pH 7.4
  

Mfp5 purification

Suspended cell pellets with 10 ml of lysis buffer per gram of wet cells and lysed it at 37°C, 220 rpm for 20 min. The cell suspension was then sonicated on ice for 30 min with a sonicator using 5 s on/ 5 s off cycles. Sonicated cell suspension was incubated at 37°C, 220 rpm, 40 min for ful dissolution of Mfp5.

The suspension was centrifuged at 20000 g for 20 min at 18°C, and the supernatant was collected to be incubated with nickel media at 37°, 30 rpm for 1 hour. Then put the mixture into the column to let it flow through. After washing with 5-10 column volumes of wash buffer, Mfp5 was eluted by 1-2 column volumes of elution buffer.

SDS-PAGE Analysis

We can find the band of Mfp5-2(Fig.2) between 20-25 kDa, suggesting the successful expression of Mfp5-2. However, an unexpected band between 15-20 kDa show in many samples, including after induction, supernatant, washing and elution. It did not result from nickel medium since the band also occurred in after induction and supernatant samples. So we inferred that Mfp5-2 might break in half during expression since it is a fusion protein made up with two repeats of Mfp5-1.

Modification of Tyrosine Residues

Only when the tyrosine residue of Mfp5 is converted into dopamine can the protein become adherent. This step requires tyrosinase catalysis, so we first replace the purified protein buffer until the environment is suitable for tyrosinase to work, and then add enzymes to complete the modification. The buffer formula and modification steps are as follows:

Buffer preparation

• Gradient buffer 1: 4 M guanidine hydrochloride, 50 mM potassium phosphates, 300 mM sodium chloride, 250 mM imidazole, pH 7.4
  
• Gradient buffer 2: 2 M guanidine hydrochloride, 50 mM potassium phosphates, 300 mM sodium chloride, 250 mM imidazole, pH 7.4
  
• Gradient buffer 3: 0.5 M guanidine hydrochloride, 50 mM potassium phosphates, 300 mM sodium chloride, 250 mM imidazole, pH 7.4
• Dialysis buffer: 100 mM sodium acetate, pH 5.5
  
• Storage buffer: 5% acetic acid
  

Mfp5 modification
Purified protein solutions were first dialyzed in gradient buffer 1, gradient buffer 2, gradient buffer 3 at 4°C for 8 hours each successively to decrease the concentration of guanidine hydrochloride.

Then protein solutions were dialyzed in dialysis buffer at 4°C for 8 hours to entirely replace its buffer. Dialyzed proteins were then diluted to a concentration of 4 mg/ml in dialysis buffer with 100 mM ascorbic acid and filtered. Tyrosinase was added to a final concentration of 250 U/ml, and the mixture was incubated at 37°C, 220 rpm for 30 min. After the reaction, the solution was filtered, and enzyme activity in the solution was quenched by adding 0.2 ml of 6 N HCl per ml of reaction. The sample was then dialyzed in 5% acetic acid and stored in 4°C.

Yield Calculation

After nickel ion affinity chromatography elution, we tested the OD value of the washing sample at 280nm and calculated the average yield. The result turned out to be 33mg per liter of bacteria solution. After modification, we tested the OD value again and found out the yield became 11mg per liter of bacteria solution or so, and the loss during modification is up to 2/3.

Mfp5-2 Adhesion Analysis

The adhesion test experiments of Mfp5-1&Mfp5-2 were carried out simultaneously as a control experiment, so the data of both Mfp5-1 and Mfp5-2 would be presented here.

To characterize as much as possible the viscosity of tyrosine enzyme-modified mefp5-1 and mefp5-2, take 20ul of the concentrated protein solution(concentration: mefp5-1=50mg/ml,mefp5-2=40mg/ml) to attach the PCR tube and the tube cover(Fig.3 top left and top right) (The PCR tube cover is trimmed and polished to a round face with a diameter of 7mm)to the center of the petri dish and perform a viscidity test after 48 hours of drying in a 37-degree oven.

Lift the petri dish at a certain height through the PCR tube and add the glass beads one by one to the petri dish until the last glass bead is added to cause the petri dish to fall off with the PCR tube(Fig.3 bottom left and bottom right). The viscosity of the added protein can be calculated by weighing the total weight of the Petri dish and glass beads.

Fig.3: Adhesion test experiment of Mfp5-1&Mfp5-2.

Each experiment was recorded with a video camera, and every moment of falling off was recorded.(Video.1) File:Adhesion test vdieo.mp4
We calculated the results by the following steps:

Fig.4:Calculation process of Mfp5 viscosity

• Compared with the viscosity of Mfp5-1 (11.05KPa/mg), the viscosity of Mfp5-2 was improved to 12.83KPa/mg, which was 16.11% higher than that of Mfp5-1.

Mechanical Test of hydrogel + Mfp5-2

To determinate the viscosity of hydrogel and hydrogel-Mussel Foot Protein mixture system.

In order to compare the viscosity and mechanical strength of Hydrogel system and Hydrogel-Mussel Foot Protein system , the same way as in the last experiment was used to measure the viscosity.

We measured the viscosity of the hydrogel alone. 50ul hydrogel (GelMA or Hydrogel-Mussel Foot Protein: GelMA+Mussel Foot Protein(mefp5-2))was used to glue the PCR tube cap with a diameter of 7mm along with the tube body to the center of the petri dish. (Fig.5a)The viscosity of GelMA was measured after curing with UV lamp for 15 seconds. (Fig.5b)

Fig.5a: Glue the PCR tube to the petri dish with GelMA or GelMA+Mussel Foot Protein

Fig.5b: Use UV light to cure GelMA or GelMA+Mussel Foot Protein.

In order to prevent the sliding of the added glass beads from changing the overall center of gravity, which results in uneven forces on the contact surface between the PCR tube and the petri dish, double-sided adhesive was affixed to the bottom of the petri dish to fix the position of the glass beads and stabilize the center of gravity(Fig.5c). Glass beads and weights were added to the petri dish one by one until the PCR tube separated from the petri dish(Fig.5d).

Fig.5c: Fill the petri dish one by one with glass beads and weights.

Fig.5d: Record the weight of glass beads when separated petri dish from PCR tube.

Calculating results

Fig.6: Calculation process of viscosity of hydrogel + Mfp5-2.

The Mussel Foot Protein +GELMA system produced a stronger viscosity than GELMA hydrogel alone,which is increased by 36.2% compared with GelMA alone and increased by about 260.8% compared to Mussel Foot Protein alone.

References

[1]Danner EW, Kan Y, Hammer MU, Israelachvili JN, Waite JH. Adhesion of mussel foot protein Mefp-5 to mica: an underwater superglue. Biochemistry. 2012 Aug 21;51(33):6511-8. doi: 10.1021/bi3002538. Epub 2012 Aug 8. PMID: 22873939; PMCID: PMC3428132.
[2]Yang B, Song J, Jiang Y, Li M, Wei J, Qin J, Peng W, Lasaosa FL, He Y, Mao H, Yang J, Gu Z. Injectable Adhesive Self-Healing Multicross-Linked Double-Network Hydrogel Facilitates Full-Thickness Skin Wound Healing. ACS Appl Mater Interfaces. 2020 Dec 30;12(52):57782-57797. doi: 10.1021/acsami.0c18948. Epub 2020 Dec 18. PMID: 33336572.
[3]Huang J, Li H, Wang Q. [Development of double-component rapid curing bioadhesive]. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2018 Dec 25;35(6):921-927. Chinese. doi: 10.7507/1001-5515.201805045. PMID: 30583318.
[4]Liang Y, Zhao X, Hu T, Han Y, Guo B. Mussel-inspired, antibacterial, conductive, antioxidant, injectable composite hydrogel wound dressing to promote the regeneration of infected skin. J Colloid Interface Sci. 2019 Nov 15;556:514-528. doi: 10.1016/j.jcis.2019.08.083. Epub 2019 Aug 24. PMID: 31473541.
[5]Yang Y, Liang Y, Chen J, Duan X, Guo B. Mussel-inspired adhesive antioxidant antibacterial hemostatic composite hydrogel wound dressing via photo-polymerization for infected skin wound healing. Bioact Mater. 2021 Jun 23;8:341-354. doi: 10.1016/j.bioactmat.2021.06.014. PMID: 34541405; PMCID: PMC8427086.
[6]Kord Forooshani P, Lee BP. Recent approaches in designing bioadhesive materials inspired by mussel adhesive protein. J Polym Sci A Polym Chem. 2017 Jan 1;55(1):9-33. doi: 10.1002/pola.28368. Epub 2016 Oct 11. PMID: 27917020; PMCID: PMC5132118.
[7]Lee BP, Messersmith PB, Israelachvili JN, Waite JH. Mussel-Inspired Adhesives and Coatings. Annu Rev Mater Res. 2011 Aug 1;41:99-132. doi: 10.1146/annurev-matsci-062910-100429. PMID: 22058660; PMCID: PMC3207216.
[8]Hwang DS, Yoo HJ, Jun JH, Moon WK, Cha HJ. Expression of functional recombinant mussel adhesive protein Mgfp-5 in Escherichia coli. Appl Environ Microbiol. 2004 Jun;70(6):3352-9. doi: 10.1128/AEM.70.6.3352-3359.2004. PMID: 15184131; PMCID: PMC427802.
[9]Kim E, Dai B, Qiao JB, Li W, Fortner JD, Zhang F. Microbially Synthesized Repeats of Mussel Foot Protein Display Enhanced Underwater Adhesion. ACS Appl Mater Interfaces. 2018 Dec 12;10(49):43003-43012. doi: 10.1021/acsami.8b14890. Epub 2018 Nov 27. PMID: 30480422.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 46
  Illegal XhoI site found at 514
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]