TY - JOUR
T1 - Improvement of Production and Isolation of Human Neuraminidase-1 in Cellulo Crystals
AU - Koiwai, Kotaro
AU - Tsukimoto, Jun
AU - Higashi, Tetsuya
AU - Mafuné, Fumitaka
AU - Miyajima, Ken
AU - Nakane, Takanori
AU - Matsugaki, Naohiro
AU - Kato, Ryuichi
AU - Sirigu, Serena
AU - Jakobi, Arjen
AU - Wilmanns, Matthias
AU - Sugahara, Michihiro
AU - Tanaka, Tomoyuki
AU - Tono, Kensuke
AU - Joti, Yasumasa
AU - Yabashi, Makina
AU - Nureki, Osamu
AU - Mizohata, Eiichi
AU - Nakatsu, Toru
AU - Nango, Eriko
AU - Iwata, So
AU - Chavas, Leonard M.G.
AU - Senda, Toshiya
AU - Itoh, Kohji
AU - Yumoto, Fumiaki
N1 - Funding Information:
We thank Rie Tanaka for administrative assistance with the SACLA SFX experiment and Elena Sablin for critical reading. TEM data analyses were supported by Naruhiko Adachi. This research was supported by the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science: PDIS) and Basis for Supporting Innovative Drug Discovery and Life Science Research from the Japan Agency for Medical Research and Development (AMED). The XFEL experiments were carried out at the BL3 of SACLA with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (proposal no. 2015B8048). The synchrotron experiment was carried out at the BL41XU of SPring-8 supported by the PDIS. This work was supported by the X-ray Free-Electron Laser Priority Strategy Program (MEXT) and JSPS KAKENHI grant number JP16K18507. We acknowledge computational support from the SACLA HPC system and Mini-K super computer system. This work was performed under the approval of the Photon Factory Program Advisory Committee (proposal no. 2016G167).
Publisher Copyright:
© 2019 American Chemical Society.
PY - 2019/11/18
Y1 - 2019/11/18
N2 - In cellulo crystallization is a developing technique to provide crystals for protein structure determination, particularly for proteins that are difficult to prepare by in vitro crystallization. This method has a key advantage: it requires neither a protein purification step nor a crystallization step. However, there is still no systematic strategy for improving the technique of in cellulo crystallization because the process occurs spontaneously. Here we report a protocol to produce and extract in cellulo crystals of human lysosomal neuraminidase-1 (NEU1) in human cultured cells. Overexpression of NEU1 protein by the retransfection of cells pretransfected with neu1-overexpressing plasmid improved the efficiency of NEU1 crystallization. Microscopic analysis revealed that NEU1 proteins were not crystallized in the lysosome but in the endoplasmic reticulum (ER). Screening of the buffer conditions used to extract crystals from cells further improved the crystal yield. The optimal pH was 7.0, which corresponds to the pH in the ER. Use of a high-yield flask with a large surface area also yielded more crystals. These optimizations enabled us to execute a serial femtosecond crystallography experiment with a sufficient number of crystals to generate a complete data set. Optimization of the in cellulo crystallization method was thus shown to be possible.
AB - In cellulo crystallization is a developing technique to provide crystals for protein structure determination, particularly for proteins that are difficult to prepare by in vitro crystallization. This method has a key advantage: it requires neither a protein purification step nor a crystallization step. However, there is still no systematic strategy for improving the technique of in cellulo crystallization because the process occurs spontaneously. Here we report a protocol to produce and extract in cellulo crystals of human lysosomal neuraminidase-1 (NEU1) in human cultured cells. Overexpression of NEU1 protein by the retransfection of cells pretransfected with neu1-overexpressing plasmid improved the efficiency of NEU1 crystallization. Microscopic analysis revealed that NEU1 proteins were not crystallized in the lysosome but in the endoplasmic reticulum (ER). Screening of the buffer conditions used to extract crystals from cells further improved the crystal yield. The optimal pH was 7.0, which corresponds to the pH in the ER. Use of a high-yield flask with a large surface area also yielded more crystals. These optimizations enabled us to execute a serial femtosecond crystallography experiment with a sufficient number of crystals to generate a complete data set. Optimization of the in cellulo crystallization method was thus shown to be possible.
KW - X-ray free electron laser
KW - endoplasmic reticulum
KW - human neuraminidase-1
KW - in cellulo crystallization
KW - protein overexpression
KW - serial femtosecond crystallography
KW - transmittance electron microscopy
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U2 - 10.1021/acsabm.9b00686
DO - 10.1021/acsabm.9b00686
M3 - Article
AN - SCOPUS:85073832529
VL - 2
SP - 4941
EP - 4952
JO - ACS Applied Bio Materials
JF - ACS Applied Bio Materials
SN - 2576-6422
IS - 11
ER -