Coding

Part:BBa_K5261003

Designed by: Yutong Gao   Group: iGEM24_HiZJU-China   (2024-09-29)


TFD-M, a monomeric protein with two binding sites for rare earth adsorption

To further enhance the rare-earth binding and rare-earth detection performance of TFD-S, we first continued to introduce amino acid targeting mutations in the inner cavity of the protein by visual inspection based on the good C2 symmetry structure of TFD-S. We used PyMol to modify the amino acid sequence. First, in order to introduce new lanthanide metal-ligand residues, we introduced the I6E/I52E/I175E/I221E mutations in TFD-S, and continued to introduce the Q154W/Q323W mutations, which resulted in the mutant TIM-FD-Mutation (TFD-M).

pET-28a(+)-TFD-M Plasmid Construction

Through several rounds of annular PCR in pET-28a(+)-TFD-S plasmid sequences within the TFD - S lead I6E/I52E/I175E/I221E/Q154W/Q323W mutation, build the E.coli expression plasmid pET-28a(+)-TFD-M (Figure 1. A). After the positive monoclone of transformed E.coli was obtained, it was sent for detection and sequencing. Through sequence alignment (Figure 1. B), the results confirmed that TFD-M gene site-specific mutation was successful.

Figure 1. A. pET-28a(+)-TFD-M plasmid B. TFD-M genetic sequence C. The sequencing results showed that the TFD-M gene was successfully inserted into the pET-28a(+) plasmid

Induced expression, purification, and SDS-PAGE process of TFD-M in Escherichia coli BL21 (DE3)

Preparation of reagents

Lysis Buffer: 300 mM NaCl, 20 mM Tris, 20 mM Imidazole, 5 mM PMSF, 100 ug/mL Lysozyme, 10 μg/mL DNase (pH = 8.0)

Wash Buffer: 25 mM HEPES, 300 mM NaCl, 20 mM Imidazole (pH 7.5)

Elution Buffer: 25 mM HEPES, 300 mM NaCl, 300 mM Imidazole (pH 7.5)

Shaking Flask Cultivations

  1. 10 µL of Escherichia coli seed solution stored in glycerol tube was collected on LB solid medium containing 50 µg/mL Kan, strewn, inverted plate, and incubated in a constant temperature incubator at 37℃ overnight.
  2. Single colonies were picked from the plate into a test tube containing 5 mL of LB liquid medium, and 5 µL Kan at a concentration of 100 mg/mL was added and incubated overnight at 37℃ on a shaker at 220 rpm.
  3. The inoculum was transferred to 100 mL LB liquid medium containing 50 µg/mL Kan, and incubated at 37℃ for 4 h until OD600 reached about 0.6-0.9, then 50 µL 0.5 M IPTG was added. Expression was induced for 16 h at 18℃.
  4. After cultivation, the bacterial solution was collected and centrifuged at 4000 rpm for 30 min. The supernatant was discarded, and the bacteria were resuspended in 10 mL Lysis Buffer and placed in an ice water bath for ultrasonic lysis. The parameters of ultrasonic cell lysis were 520 W, 3 s ultrasonic work, 7 s interval, and 150 cycles. The cytoclastic fluid was centrifuged at 4000 rpm for 20 min, and the supernatant containing the target protein was collected.

Purification

pET28a (+)-TFD-M has a 6×His tag added to the C terminus, which can specifically bind to Ni ions, so the Ni-NTA affinity column was used for protein purification experiments.

  1. The Ni-NTA packing column was cleaned with 5 times the column volume of deionized water and then equilibrated with 5 times the column volume of Lysis Buffer.
  2. The supernatant obtained by cell lysis was further filtered by 0.22 µm filter membrane to avoid contaminating the packed column, and the sample was repeated 5 times to fully combine the protein with the Ni column.
  3. Wash Buffer with 10 times the column volume to remove impurities and wash out the residual miscellaneous proteins in the column.
  4. Finally, the target protein was eluted with 3 times the column volume Elution Buffer and the elution solution was collected.
  5. The column material was eluted with 5x column volume Elution Buffer and cleaned with 5x column volume deionized water, and the Ni-NTA affinity column was preserved with 20% alcohol.
  6. Pour the protein eluent into a Millipore ultrafiltration tube with a molecular weight cut-off of 10 kDa, centrifuge at 4000 rpm for 20 min, add deionized water to the original volume, then centrifuge, repeat the operation for 3 times, absorb the protein concentrate into a 1.5 mL EP tube, and store at 4℃.

SDS-PAGE Validation

  1. Protein sample preparation: 40 µL protein solution was sucked and mixed with 10 µL SDS-PAGE protein loading buffer, heated at 99℃ for 10 min, and then loaded after cooling.
  2. Electrophoresis: MOPS buffer was added to the electrophoresis tank, and 5 µL protein marker and 10 µL protein sample were added to the loading well, respectively. The voltage was set at 160 V, and the electrophoresis time was 1 h.
  3. Staining: After the end of electrophoresis, the protein glue was removed, the dye Coomassie Brilliant blue R250 was added to immerse it, and the staining was shaken for 30 min.
  4. Decolorization: Dip the dyed protein glue into water and shake it overnight to decolorize until clear protein bands are seen.
  5. Imaging: Image acquisition was performed in a gel imager.

Subsequently, we expressed and purified TFD-M protein in Escherichia coli BL21(DE3), and verified by SDS-PAGE, the mutant protein TFD-M was successfully obtained (theoretical band 37.67kDa) (Figure 2).

Figure 2. SDS-PAGE and Coomassie bright blue staining of TFD-M (Note: TFD-M-1 and TFD-M-2 are two parallel samples. Lane 1: Initial supernatant, Lane 2: precipitation after centrifugation, Lane 3: buffer elute, Lane 4: Dilute imidazole elute, Lane 5: concentrated imidazole elute, Lane 6: protein concentrate obtained after ultrafiltration)

Computer Visualization and Simulations

AlphaFold3 Prediction

We used PyMol to modify the amino acid sequence of TIM-FD-Mutation (TFD-M) and gave the sequence to AlphaFold3 for prediction.The results showed that the average distance between the 8 active oxygen atoms of TFD-M and terbium ions was 2.41 Å (Figure 3).

Figure 3. DNA manipulating methods from the literatures

MD Simulation Analysis

MD simulation was also performed using CHARMM36 protein Force Field 45 in the GROMACS2022.3 package 46. The force constant of the harmonic bias potential is 1000 kJ∙mol-1∙nm-2. Set the 10 Å cuboid box where the edge is widest from the protein, use the TIP3P water model in it, and add sodium ions to the solution to maintain the neutrality of the system. Eleven TFD-S-X(III) ( X(III) means Tb(III) or La(III) or Lu(III) ) systems were simulated at 300 K. To begin with, the system is minimized by 50,000 steps using the steepest descent algorithm. In the pre-balancing phase, the system is gradually heated to 300 K at 1 atm using a 200ps NVT set and 200ps NPT set. A 50ns production run followed to collect the balanced configuration for each 2ps interval. A speed scaling thermostat 49 with a time constant equal to 0.1ps was used to keep the temperature constant throughout the simulation. To maintain the pressure, the Berendsen pressure coupler was used in the NPT pre-balance run and the Parrinello-Rahman pressure coupler was used in the production run, with the pressure time constant and isothermal compression rate set at 2ps and 4.5×10-5 bar-1, respectively. In the whole simulation process, the integral time step of the motion equation is 2fs. The cut-off value for the non-bonding interaction is 12Å. The particle grid Ewald algorithm 53 is used to calculate long range electrostatic interactions. [3] We tested the binding of TFD-M with Lu(III), and the final MD results of the three ions, Tb(III), La(III), Lu(III),are shown as follows (Figure 4).

Figure 4. Schematic diagram of TFD-M combined with Tb(III), La(III), Lu(III), molecular dynamics simulation process, and kinetic curve. The ordinate of the RMSD curve is the root-mean-square deviation, indicating the distance the system moves.

Figure 5. the interaction between Tb ions and TFD-M.

Figure 6.the interaction between La ions and TFD-M

Figure 7. the interaction between Lu ions and TFD-M.

Protein Characterization

Binding ability of TFD-M to different lanthanide metal ions

Firstly, the binding of TFD-M and Tb(III) was characterized by the terbium luminescence mechanism sensitized by antenna effect. The 10 μM TbCl3 and 5 μM TFD proteins were pre-incubated in Tris-HCl buffer with pH=7.5 for 1 hour, then added into 96-well plates. The excitation wavelength was set at 280nm, the detection emission wavelength ranged from 520nm to 570nm, and the step length was 5nm using a time-resolved fluorescence mode (TRF). Detection of terbium emission enhanced by tryptophan. Subsequently, in order to detect the adsorption capacity of proteins for different rare earth ions, Tb(III) in the incubated proteins was replaced with other lanthanide ions (except radioactive element Pm(III)), and the luminescence intensity was detected every 30min, and the decay curve of the Tb(III) fluorescence value enhanced by tryptophan was measured over time (Figure 5).

Figure 8. Tb（III） fluorescence decay curves of TFD-M-Tb(III) after substitution by different rare earth ions

Measurement of binding capacity of lanthanide metal elements by TFD-M under different pH conditions

Rare earth ore bioleach is usually acidic, so we need to further examine the difference in the binding ability of TFD protein to lanthanide metals under acidic conditions. We selected Tb(III) ion for adsorption experiment. We used 1M hydrochloric acid solution with pH=5.0/5.5/6.0; Configure a Tris-HCl buffer with a pH of 6.5/7.0/7.5/8.0/8.5 100 mM using a Tris-HCl and sodium hydroxide solution with a pH of 6.8. Each well was added in accordance with the incubation system of 200 μL, and the test was started immediately with an enzyme-labeled instrument (the parameters were the same as those of the adsorption displacement experiment) at a time interval of 15min. The curves of the adsorption reaction of TFD-M with Tb(III) at different pH were obtained. For TFD-M (Figure 6), the terbium luminescence intensity was at a low level at pH < 6.0 or pH > 7.0, and the increasing trend with time was not obvious, indicating that TFD-M has almost no adsorption capacity for Tb(III) in the pH < 6.0 or pH > 7.0 range. When the ambient pH = 6.5, the terbium luminescence intensity also reached the maximum, and the luminescence intensity increased with time, indicating that TFD-M has the ability to adsorb Tb(III) at pH = 6.5, but the luminescence intensity was much smaller than the terbium luminescence produced by the binding of TFD-S to Tb(III) under the same ambient conditions, which indicated that our modified mutant TFD-M was not able to achieve the purpose of increasing the binding site of Tb(III) in the protein. ) binding site within the protein. By visual inspection of the TFD-M rare-earth binding site, we speculate that the possible reason is that the inner cavity of the TFD-M protein is small and the barrel cannot accommodate two rare-earth ions, and the two Tb(III) ions lead to an unstable binding under the action of repulsive forces.

Figure 9. Adsorption of Tb (III) by TFD-M in the pH range of 5.0-8.5

References

[1]Caldwell, Shane J., et al. Tight and specific lanthanide binding in a de novo TIM barrel with a large internal cavity designed by symmetric domain fusion. Proceedings of the National Academy of Sciences. 2020, 117(48): 30362-30369.

[2]Martin, Langdon J., and Barbara Imperiali. The best and the brightest: Exploiting tryptophan-sensitized Tb 3+ luminescence to engineer lanthanide-binding tags. Peptide libraries: methods and protocols. 2015: 201-220.

[3] Tutorials and Webinars — GROMACS webpage https://www.gromacs.org documentation



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 572
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 310
  • 1000
    COMPATIBLE WITH RFC[1000]


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