Part:BBa_K5261000

TFD-EE , a homodimeric protein for rare earth adsorption

The TFD-EE protein adsorbs lanthanide metal ions in solution and quantitatively detects lanthanide binding using a tryptophan-enhanced ‘antenna effect’ luminescence mechanism.

It is a dimeric protein with C2 symmetry, incorporating a TIM barrel fold and a ferredoxin fold, with a large inner cavity and a unique structure that provides lanthanide coordination using glutamate residues.

Different lanthanide metals have different fluorescence emission wavelengths, but due to the low extinction coefficient of lanthanide metals, they cannot be directly excited, and the indole ring on the surrounding tryptophan can absorb the external energy and then transfer it to the lanthanide metal ions to be excited to produce fluorescence.

AlphaFold3 Prediction

TFD-EE is a C2-symmetric dimer protein capable of adsorbing rare earth ions, with a total of eight β-sheet in the barrel, and four C4-symmetric glutamates located on four separate β-sheet (Figure 1). To test for protein binding, Shane J. Caldwell et al.[1] introduced a tryptophan (N6W mutation) as a sensitizer antenna effect to detect Tb(III) luminescence [2]. That is, when the protein binds Tb(III), the excitation wavelength of 280 nm is given, and the indole ring on tryptophan can absorb energy and transfer it to Tb(III), thus making Tb(III) emit characteristic fluorescence, and the adsorption of Tb(III) can be quantitatively detected according to the fluorescence intensity.

Figure 1. Combination of TFD-EE and Tb(III)

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-S 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 3 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.

Finally,we expressed and purified TFD-EE protein in Escherichia coli BL21(DE3), which was verified by SDS-PAGE, indicating that the TFD-EE protein was successfully obtained (theoretical band 18.75kDa) (Figure 2).

Figure 2. SDS-PAGE and Coomassie bright blue staining of TFD-EE(Note: 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)

Reference

[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

Sequence and Features

Assembly Compatibility:

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  Illegal AgeI site found at 394
  
• 1000
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