Architecture and mechanism of the shelterin complex

2021 Fiscal Year Research-status Report

• Project/Area Number: 21K20645
• Research Institution: Okinawa Institute of Science and Technology Graduate University
• Principal Investigator: AYALA・HERNANDEZ Rafael 沖縄科学技術大学院大学, 生体分子電子顕微鏡解析ユニット, ポストドクトラルスカラー (00912601)
• Project Period (FY): 2021-08-30 – 2023-03-31
• Keywords: cryoEM / telomeres / chromatin

Outline of Annual Research Achievements

Sequence of all 6 genes encoding the components of the human shelterin complex have been codon optimized for expression in insect cells and synthesized as synthetic genes. The genes have been cloned under the control of baculoviral promoter and terminator, cloned into a single pFBDM vector and transferred to a bacmid backbone. Backed has been used to transfect Sf9 insect cells to generate baculoviruses carrying human shelterin genes. The baculoviruses have been used to infect large volume of Sf9 cells to express shelterin, which has been purified through affinity chromatography. The purified sample has been analyzed by negative staining microscopy to verify that the sample is in good state.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

The multi-gene human shelterin complex has been successfully expressed and purified recombinantly in a baculovirus-insect cell expression system, and the negative stain micrographs look promising and show that individual complexes can be distinguished. We are now in the process of preparing grids for cryo-EM and collect cryo-EM data, which will lead to the high-resolution structure of shelterin.

Strategy for Future Research Activity

The purification buffer contained 10% glycerol. This is not a problem for negative stain micrographs, but it is not compatible with cryo-EM since it largely reduces contrast. Therefore, the next step will be to remove glycerol from the sample by means of dialysis. After that, the dialyzed sample will be used to prepare cryo-EM grids, which will be imaged. If the sample is found to be in good state for cryo-EM, a large dataset will be collected and processed. If aggregation is observed, the sample will be subjected to an additional purification step by means of gel filtration before preparing cryo-EM grids again.

Causes of Carryover

The incurring amount will be used to cover costs of larger expression volumes and to try a different strain of insect cells (High Five), which require a different type of medium and glutamine supplements. Additionally, it will be used to cover the costs of different type of cryo-EM grids to find the most appropriate ones for this complex. Finally, it will also be used to cover the costs of gel filtration column if required to increase even more sample purity.