Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3′ end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.
1. Expression of recombinant TcTERT
2. Purification of TcTERT
3. Tribolium castaneum beetle culture
4. T. castaneum Total RNA isolation
5. In vitro reconstitution of Tribolium castaneum telomerase
6. Telomerase repeat amplification protocol (TRAP) assays
7. Representative Results:
An example of the purified TcTERT after size exclusion chromatography is shown in Figure 2. The protein concentrated after purification is run on a 12% SDS-polyacrylamide gel and is shown to be more than 99% pure (Figure 2A). The size exclusion (S200) chromatogram is also provided (Figure 2B) to demonstrate the purity and lack of aggregates from the purified protein sample. A yield of 5 mg protein after all purification steps have been completed is usually obtained although the final yield can vary.
A representative TRAP assay for the reconstituted T. castaneum telomerase is shown in Figure 3 as previously reported by Mitchell et al.12. The step-wise ladder of telomerase products can be seen in lane 1, however they are absent in both the RNase treated telomerase and the TERT active site mutant (D251A) samples in lanes 2 and 3 respectively.
Figure 1. Flow chart of the steps involved in the reconstitution of the recombinant TcTERT. First, the recombinant TcTERT is over-expressed in E. coli. The protein is then purified by nickel, cation exchange, and size exclusion chromatography. The T. castaneum beetles are cultured in house and their larvae are used to isolate the total RNA. The purified TcTERT and the total RNA are then mixed to reconstitute the telomerase RNP complex, and a subsequent TRAP assay is used to confirm the RNP complex is an active telomerase.
Figure 2. Size exclusion chromatogram and SDS PAGE analysis of TcTERT protein. (A) 12% SDS-polyacrylamide gel of the TcTERT protein after size exclusion chromatography. The TcTERT protein (70 kDa) migrates faster on the SDS PAGE gel because it has a high isoelectric point (pI – 9.8). (B) Size exclusion chromatogram (Superdex S200) of the TcTERT protein.
Figure 3. TRAP assays of the in vitro reconstituted Tribolium castaneum telomerase adapted from Mitchell et al.12. Lane 1: wild type TcTERT and total RNA isolated from beetle larvae. Lane 2: wild type TcTERT and RNase treated larvae total RNA and cell extracts. Lane 3: active site mutant TcTERT (D251A) and beetle larvae total larvae RNA.
The method presented here allows for the production of large amounts of the catalytic subunit of T. castaneum telomerase TERT in soluble, active form for structural and biochemical studies. The method of TcTERT over-expression is sensitive to subtle changes in temperature or cell density from those stated above and can dramatically affect the levels of protein expression. Specifically, we have found that inducing the cells for protein expression before the optical density reaches 0.5 at 600 nm, or lowering the temperature to 30°C before induction, leads to a significant reduction in the protein yield.
In terms of purification, the TcTERT protein has a basic isoelectric point (pI – 9.8) and so its ability to bind tightly to the cation-exchange column can be exploited to yield a pure protein sample by loading and washing the sample with high salt concentrations. If a protein of interest were to have a low isoelectric point (pI < 5) this protocol could be adapted by substituting the cation-exchange for an anion-exchange column, and re-optimizing the wash conditions to achieve the best protein purity.
In assembling the enzyme, the method described here is only a partial reconstitution of the telomerase holoenzyme because it does not include any telomerase associated proteins. Although these components are not present, the reconstituted telomerase consisting of the recombinant TcTERT and total RNA from T. castaneum larvae is active as shown in Figure 3. The unique ability for telomerase to add multiple identical repeats to the ends of chromosomes, a process known as Repeat Addition Processivity, can be visualized by the ladder of products in the TRAP gel.
The authors have nothing to disclose.
The research presented here was supported by the Pennsylvania Department of Health, The Ellison Medical, The V and The Emerald Foundations.
Name of Reagent | Company | Catalog Number |
---|---|---|
Rosetta(DE3)plysS Cells | Novagen | 70956 |
2YT Broth | Teknova | Y0215 |
IPTG | Gold Biotechnology | I2481C |
MISONIX Sonicator 3000 | Qsonica, LLC. | |
ÄKTA Purifier FPLC | GE Life Sciences | |
Ni-NTA Superflow Resin | Qiagen | 30410 |
Amicon Ultra-15 Centrifugal Filter Device | Millipore | UFC903008 |
POROS 50 HS Strong Cation Exchange Packing | Applied Biosystems | 1-3359-06 |
POROS 50 HQ Strong Cation Exchange Packing | Applied Biosystems | 1-2559-06 |
Superdex 200 10/300 Size-Exclusion Column | GE Life Sciences | 17-5175-01 |
Phenol: Chloroform: Isoamyl 25:24:1 with 10mM Tris, pH 8, 1mM EDTA | Sigma | P3803-100mL |
RNaseZap | Ambion | AM9780 |
Recombinant Rnasin Ribonuclease Inhibitor | Promega | N251B |
RNeasy Mini Kit | Qiagen | 74104 |
DNA oligonucleotides | Integrated DNA Technologies |