Cell-free protein crystallization for nanocrystal structure determination

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 fo...

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Bibliographic Details
Published in:Scientific reports 2022-10, Vol.12 (1), p.16031-16031, Article 16031
Main Authors: Abe, Satoshi, Tanaka, Junko, Kojima, Mariko, Kanamaru, Shuji, Hirata, Kunio, Yamashita, Keitaro, Kobayashi, Ayako, Ueno, Takafumi
Format: Article
Language:English
Subjects:
631/1647/2258/1266
631/535/1266/1265
631/553/552
639/638/541/961
Crystallization
Crystals
Dialysis
Humanities and Social Sciences
multidisciplinary
Polyhedra
Protein biosynthesis
Protein purification
Protein structure
Protein synthesis
Proteins
Reagents
Science
Science (multidisciplinary)
Structural analysis
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Summary: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.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-022-19681-9