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Publication Abstract from PubMed

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 A. Furthermore, nanocrystals were synthesized at a reaction scale of only 20 muL using the dialysis method, enabling structural analysis at a resolution of 1.95 A. 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 A 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.

Cell-free protein crystallization for nanocrystal structure determination.,Abe S, Tanaka J, Kojima M, Kanamaru S, Hirata K, Yamashita K, Kobayashi A, Ueno T Sci Rep. 2022 Oct 3;12(1):16031. doi: 10.1038/s41598-022-19681-9. PMID:36192567^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.