Improvement of Production and Isolation of Human Neuraminidase-1 in Cellulo Crystals

Kotaro Koiwai, Jun Tsukimoto, Tetsuya Higashi, Fumitaka Mafuné, Ken Miyajima, Takanori Nakane, Naohiro Matsugaki, Ryuichi Kato, Arjen Jakobi, More Authors

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Abstract

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.

Original languageEnglish
Pages (from-to)4941-4952
Number of pages12
JournalACS Applied Bio Materials
Volume2
Issue number11
DOIs
Publication statusPublished - 2019

Bibliographical note

Accepted Author Manuscript

Keywords

  • endoplasmic reticulum
  • human neuraminidase-1
  • In cellulo crystallization
  • protein overexpression
  • serial femtosecond crystallography
  • transmittance electron microscopy
  • X-ray free electron laser

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