expression and purification of telomeric dna-binding protein trf2 using sumo fusion tag

Studying TRF1/2 Interactions with Telomeric DNA Using Single-Molecule Assay

Telomeres are protective structures at the ends of chromosomes1,2,3. Telomere erosion during cell division leads to cell senescence and aging, while abnormal elongation of telomeres contributes to cancer4,5. For telomeres to function properly, their replication, elongation, and damage responses must be highly regulated6,7,8. Shelterin, composed of six subunits, plays a central role in telomere protection9,10,11. A deeper understanding of telomeres will provide valuable insights into telomere biology.
TRF1 and TRF2, core subunits of shelterin, are telomeric binding proteins12,13. Both TRF1 and TRF2 bind to the repetitive DNA motif TTAGGG in telomeres via their Myb domains14. They form dimers through their shared TRFH domains, which allow them to encircle telomeric double-stranded DNA and to recruit telomeric proteins15,16,17,18,19. TRF2 is particularly important for the formation of telomeric D-loops and T-loops20,21. Due to their crucial roles in telomere protection, TRF1 and TRF2 have emerged as potential anti-cancer drug targets22,23,24,25.
Significant efforts have been made to investigate the protein-DNA interactions at telomeres. Biochemical methods such as Electrophoretic Mobility Shift Assay (EMSA) and Surface Plasmon Resonance (SPR) have been used to examine binding affinities20,26. Numerous structures of telomeric binding proteins complexed with DNA have been elucidated using cryo-electron microscopy (cryo-EM), X-ray crystallography, and nuclear magnetic resonance (NMR)27,28,29. Super-resolution imaging techniques like stochastic optical reconstruction microscopy (STORM) have revealed TRF2-dependent T-loop formation21. Recently, nanopore sequencing has been developed to profile telomeric sequences4,30,31. These structural insights have greatly enhanced our understanding of telomeric protein-DNA interactions. To further explore the dynamics of telomeric protein-DNA interactions, the development of new technologies is essential.
Single-molecule tools are powerful techniques for exploring protein-DNA interactions at telomere32,33,34. Single-molecule mechanical methods, such as magnetic tweezers, optical tweezers and AFM, have been employed to investigate TRF2-dependent DNA distortion, reveal TRF2-mediated columnar stacking of human telomeric chromatin and observe processive telomerase catalysis, among other applications35,36,37,38,39,40. These methods are particularly useful for probing topological conformations and the kinetics of protein-DNA association and dissociation.
However, the preparation of single-molecule constructs with telomeric repetitive motifs still presents challenges, which limits studies using single-molecule mechanical methods. To address this limitation, we have developed a single-molecule mechanical method to study global protein-DNA interactions on full-length human telomeres41. This method directly extracts telomeric DNA from human cells, circumventing the laborious preparation of artificial telomeric DNA. It facilitates the investigation of kinetic processes on long native telomeres spanning several kilobases.
In this protocol, we provide a detailed description of the steps for probing telomeric protein-DNA interactions using magnetic tweezers, a popular single-molecule mechanical tool42,43,44. We demonstrate how to express and purify telomeric proteins, using TRF2 as an example, and how to prepare telomeric DNA from human cells. Additionally, we show how to set up a single-molecule assay on magnetic tweezers to study telomeric protein-DNA interactions, and we cover the subsequent data analysis of single-molecule experiments. This protocol will benefit researchers in the field of telomere biology and telomere-targeted drug discovery.

Author Spotlight: Advanced Single-Molecule Techniques for Investigating Telomeric Protein-DNA Interactions

Analyzing Telomeric Protein-DNA Interactions Using Single-Molecule Magnetic Tweezers

Single-molecule methods have been used to study telomeric protein-DNA interaction. However, preparing single molecule constructs with the telomeric repetitive motifs remains a challenging task. In this protocol, we outline the steps for expressing and purifying TRF2 protein, preparing telomeric DNA, setting up single-molecule mechanical assays, and analyzing the resulting data.Single-molecule tools are powerful techniques for exploring telomeric protein-DNA interactions. Single-molecule mechanical methods, such as magnetic tweezers, optical tweezers, and AFM have been used to investigate TRF2-dependent DNA distortion, reveal TRF2-mediated columnar stacking of human telomeric chromatin, and observe the presence of telomerase catalysis among other applications. We investigated telomeric DNA-protein interactions using single molecular magnetic tweezers, enabling precise measurements of changes in extension and the durations of protein-DNA interactions under applied forces.From these measurements, we derived the dissociation kinetics of the telomeric DNA-protein complexes. Furthermore, the formation of loops within these complexes was revealed. Numerous structures of telomeric-binding proteins in complex with DNA have been resolved using cryo-EM, X-ray crystallography, and NMR.These structural insights have advanced our understanding of telomeric protein-DNA interactions. To further investigate the dynamics of these interactions, we have developed a single-molecule mechanical methods specifically for studying telomeric protein-DNA interactions. Single-molecule tools are powerful techniques for investigating protein-DNA interactions at telomeres.These methods are especially valuable for topological conformations and analyzing the kinetics of protein-DNA association and dissociation.

Expression and Purification of Telomeric DNA-Binding Protein TRF2 Using SUMO Fusion Tag

To begin the transformation of Escherichia coli BL21 DE3 cells, thaw 50 microliter aliquot of competent cell suspension on ice. Add one microliter of DNA of pET 28a Sumo TRF2 plasmid to the competent cell suspension, and gently swirl the tube to mix the suspension. After transformation, spread 100 microliters of the cell suspension on a luria bertani, or LB, agar plate containing 50 micrograms per milliliter kanamycin.Incubate the cells overnight at 37 degrees Celsius. The next day, pick a colony of the transformed cells, and culture it in five milliliters of LB medium, supplemented with 50 micrograms per milliliter kanamycin. Incubate for 18 hours at 37 degrees Celsius, with shaking at 220 RPM.After incubation, induce the culture with one millimolar IPTG as a final concentration. Incubate at 20 degrees Celsius for 17 hours to promote protein expression. Load the crude protein extracts, along with the loading buffer, onto an SDS-PAGE gel.Run the samples initially at 100 volts for 30 minutes, followed by 120 volts for 50 minutes in a tris-glycine buffer. Then, stain with Coomassie blue to visualize protein expression. For protein purification, centrifuge the crude protein extract at 38, 000 G for 40 minutes at four degrees Celsius, and filter the supernatant through a 0.22 micrometer syringe filter.Load the filtered supernatant onto the pre-equilibrated nickel column in the binding buffer. Wash the column with binding buffer and elute the protein with the prepared imidazole gradient, collecting 12 fractions of one milliliter each. Add glycerol to a final concentration of 50%to the concentrated and buffer exchanged protein for storage.The expression of TRF2 in Escherichia coli was successfully demonstrated, as shown by SDS-PAGE, where distinct bands corresponding to TRF2 appeared before and after induction.

expression-purification-telomeric-dna-binding-protein-trf2-using-sumo

Research

JoVE Journal

Biochemistry

30 Views. To begin the transformation of Escherichia coli BL21 DE3 cells, thaw 50 microliter aliquot of competent cell suspension on ice. Add one microliter of DNA of pET 28a Sumo TRF2 plasmid to the competent cell suspension, and gently swirl the tube to mix the suspension. After transformation, spread 100 microliters of the cell suspension on a luria bertani, or LB, agar plate containing 50 micrograms per milliliter kanamycin.Incubate the cells overnight at 37 degrees Celsius. The next day, pi...