Cell-free protein crystallization for nanocrystal structure determination.

Autor: Abe S; School of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta-cho 4259, Midori-ku, Yokohama, 226-8501, Japan. saabe@bio.titech.ac.jp., Tanaka J; School of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta-cho 4259, Midori-ku, Yokohama, 226-8501, Japan., Kojima M; School of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta-cho 4259, Midori-ku, Yokohama, 226-8501, Japan., Kanamaru S; School of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta-cho 4259, Midori-ku, Yokohama, 226-8501, Japan., Hirata K; SR Life Science Instrumentation Unit, RIKEN/SPring-8 Center, 1-1-1, Kouto, Sayo-cho, Sayo-gun, Hyogo, 679-5148, Japan., Yamashita K; SR Life Science Instrumentation Unit, RIKEN/SPring-8 Center, 1-1-1, Kouto, Sayo-cho, Sayo-gun, Hyogo, 679-5148, Japan.; MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK., Kobayashi A; School of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta-cho 4259, Midori-ku, Yokohama, 226-8501, Japan., Ueno T; School of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta-cho 4259, Midori-ku, Yokohama, 226-8501, Japan. tueno@bio.titech.ac.jp.; International Research Frontiers Initiative (IRFI), Tokyo Institute of Technology, Nagatsuta-cho 4259, Midori-ku, Yokohama, 226-8501, Japan. tueno@bio.titech.ac.jp.
Jazyk: angličtina
Zdroj: Scientific reports [Sci Rep] 2022 Oct 03; Vol. 12 (1), pp. 16031. Date of Electronic Publication: 2022 Oct 03.
DOI: 10.1038/s41598-022-19681-9
Abstrakt: In-cell protein crystallization (ICPC) has been investigated as a technique to support the advancement of structural biology because it does not require protein purification and a complicated crystallization process. However, only a few protein structures have been reported because these crystals formed incidentally in living cells and are insufficient in size and quality for structure analysis. Here, we have developed a cell-free protein crystallization (CFPC) method, which involves direct protein crystallization using cell-free protein synthesis. We have succeeded in crystallization and structure determination of nano-sized polyhedra crystal (PhC) at a high resolution of 1.80 Å. Furthermore, nanocrystals were synthesized at a reaction scale of only 20 μL using the dialysis method, enabling structural analysis at a resolution of 1.95 Å. To further demonstrate the potential of CFPC, we attempted to determine the structure of crystalline inclusion protein A (CipA), whose structure had not yet been determined. We added chemical reagents as a twinning inhibitor to the CFPC solution, which enabled us to determine the structure of CipA at 2.11 Å resolution. This technology greatly expands the high-throughput structure determination method of unstable, low-yield, fusion, and substrate-biding proteins that have been difficult to analyze with conventional methods.
(© 2022. The Author(s).)
Databáze: MEDLINE
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