Novel Device and Strategy for Growing Large, High-Quality Protein Crystals by Controlling Crystallization Conditions

Neutron diffraction experiments are informative for determining the locations of hydrogen atoms in protein molecules; however, much larger crystals are needed than those required for X-ray diffraction. Thus, additional techniques are required to grow larger crystals. Here, a unique crystallization d...

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Main Authors: Naoki Tanigawa, Sachiko Takahashi, Bin Yan, Masayuki Kamo, Naoki Furubayashi, Koji Kubota, Koji Inaka, Hiroaki Tanaka
Format: Article
Language:English
Published: MDPI AG 2021-10-01
Series:Crystals
Subjects:
Online Access:https://www.mdpi.com/2073-4352/11/11/1311
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author Naoki Tanigawa
Sachiko Takahashi
Bin Yan
Masayuki Kamo
Naoki Furubayashi
Koji Kubota
Koji Inaka
Hiroaki Tanaka
author_facet Naoki Tanigawa
Sachiko Takahashi
Bin Yan
Masayuki Kamo
Naoki Furubayashi
Koji Kubota
Koji Inaka
Hiroaki Tanaka
author_sort Naoki Tanigawa
collection DOAJ
description Neutron diffraction experiments are informative for determining the locations of hydrogen atoms in protein molecules; however, much larger crystals are needed than those required for X-ray diffraction. Thus, additional techniques are required to grow larger crystals. Here, a unique crystallization device and strategy for growing large protein crystals are introduced. The device uses two micropumps to control crystal growth by altering the precipitant concentration and regulating the pinpoint injection of dry air flow to the crystallization cell. Furthermore, the crystal growth can be observed in real time. Preliminary microbatch crystallization experiments at various concentration ranges of polyethylene glycol (PEG) 4000 and sodium chloride were first performed to elucidate optimized crystallization conditions. Based on these results, a device to precisely control the sodium chloride and PEG concentrations and the supply of dry air to the crystallization cell was used, and 1.8 mm lysozyme and 1.5 mm alpha-amylase crystals with good reproducibility were obtained. X-ray data sets of both crystals were collected at room temperature at BL2S1 of the Aichi Synchrotron Radiation Center and confirmed that these crystals were of high quality. Therefore, this crystallization device and strategy were effective for growing large, high-quality protein crystals.
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spelling doaj.art-aea33564518b4825970c771111e3b5fc2023-11-22T22:57:45ZengMDPI AGCrystals2073-43522021-10-011111131110.3390/cryst11111311Novel Device and Strategy for Growing Large, High-Quality Protein Crystals by Controlling Crystallization ConditionsNaoki Tanigawa0Sachiko Takahashi1Bin Yan2Masayuki Kamo3Naoki Furubayashi4Koji Kubota5Koji Inaka6Hiroaki Tanaka7Chiyoda Corporation, 3-13 Moriya-cho, Kanagawa-ku, Kanagawa, Yokohama 221-0022, JapanConfocal Science Incorporated, 5-14-15 Fukasawa, Setagaya-ku, Tokyo 158-0081, JapanConfocal Science Incorporated, 5-14-15 Fukasawa, Setagaya-ku, Tokyo 158-0081, JapanMaruwa Foods and Biosciences Incorporated, 170-1 Tsutsui-cho, Yamatokoriyama, Nara 639-1123, JapanMaruwa Foods and Biosciences Incorporated, 170-1 Tsutsui-cho, Yamatokoriyama, Nara 639-1123, JapanChiyoda Corporation, 3-13 Moriya-cho, Kanagawa-ku, Kanagawa, Yokohama 221-0022, JapanMaruwa Foods and Biosciences Incorporated, 170-1 Tsutsui-cho, Yamatokoriyama, Nara 639-1123, JapanConfocal Science Incorporated, 5-14-15 Fukasawa, Setagaya-ku, Tokyo 158-0081, JapanNeutron diffraction experiments are informative for determining the locations of hydrogen atoms in protein molecules; however, much larger crystals are needed than those required for X-ray diffraction. Thus, additional techniques are required to grow larger crystals. Here, a unique crystallization device and strategy for growing large protein crystals are introduced. The device uses two micropumps to control crystal growth by altering the precipitant concentration and regulating the pinpoint injection of dry air flow to the crystallization cell. Furthermore, the crystal growth can be observed in real time. Preliminary microbatch crystallization experiments at various concentration ranges of polyethylene glycol (PEG) 4000 and sodium chloride were first performed to elucidate optimized crystallization conditions. Based on these results, a device to precisely control the sodium chloride and PEG concentrations and the supply of dry air to the crystallization cell was used, and 1.8 mm lysozyme and 1.5 mm alpha-amylase crystals with good reproducibility were obtained. X-ray data sets of both crystals were collected at room temperature at BL2S1 of the Aichi Synchrotron Radiation Center and confirmed that these crystals were of high quality. Therefore, this crystallization device and strategy were effective for growing large, high-quality protein crystals.https://www.mdpi.com/2073-4352/11/11/1311neutron diffractionlarge crystalreservoir controlreal-time observation
spellingShingle Naoki Tanigawa
Sachiko Takahashi
Bin Yan
Masayuki Kamo
Naoki Furubayashi
Koji Kubota
Koji Inaka
Hiroaki Tanaka
Novel Device and Strategy for Growing Large, High-Quality Protein Crystals by Controlling Crystallization Conditions
Crystals
neutron diffraction
large crystal
reservoir control
real-time observation
title Novel Device and Strategy for Growing Large, High-Quality Protein Crystals by Controlling Crystallization Conditions
title_full Novel Device and Strategy for Growing Large, High-Quality Protein Crystals by Controlling Crystallization Conditions
title_fullStr Novel Device and Strategy for Growing Large, High-Quality Protein Crystals by Controlling Crystallization Conditions
title_full_unstemmed Novel Device and Strategy for Growing Large, High-Quality Protein Crystals by Controlling Crystallization Conditions
title_short Novel Device and Strategy for Growing Large, High-Quality Protein Crystals by Controlling Crystallization Conditions
title_sort novel device and strategy for growing large high quality protein crystals by controlling crystallization conditions
topic neutron diffraction
large crystal
reservoir control
real-time observation
url https://www.mdpi.com/2073-4352/11/11/1311
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