Journal Article

PUBDB-2025-04364

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Intracellular protein crystallization in living insect cells

Schönherr, R. ; Eichler, N. ; Sornaly, F. A. ; Boger, J. ; Frevert, A. M. ; Lahey-Rudolph, J. M. ; Meyer, H. ; Weymar, L. ; Redecke, L. (Corresponding author)Extern*DESY*

2025
Wiley Hoboken, NJ

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Please use a persistent id in citations: doi:10.1002/2211-5463.70020  doi:10.3204/PUBDB-2025-04364

Abstract: Crystallization of recombinant proteins in living cells is an emerging approach complementing conventional crystallization techniques. Homogeneous microcrystals well suited for serial diffraction experiments at X-ray free-electron lasers and synchrotron sources can be produced in a quasi-native environment, without the need for target protein purification. Several protein structures have already been solved; however, exploiting the full potential of this approach requires a systematic and versatile screening strategy for intracellular crystal growth. Recently, we published InCellCryst, a streamlined pipeline for producing microcrystals within living insect cells. Here, we present the detailed protocol, including optimized target gene expression using a baculovirus vector system, crystal formation, detection, and serial X-ray diffraction directly in the cells. The specific environment within the different cellular compartments acts as a screening parameter to maximize the probability of crystal growth. If successful, diffraction data can be collected 24 days after the start of target gene cloning.

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Contributing Institute(s):

1. FS-Photon Science (FS-PS)
2. Universität zu Lübeck (U Lübeck)

Research Program(s):

1. 633 - Life Sciences – Building Blocks of Life: Structure and Function (POF4-633) (POF4-633)
2. DFG project G:(GEPRIS)49701054 - EXC 306: Entzündungen an Grenzflächen (49701054) (49701054)

Experiment(s):

1. No specific instrument

Appears in the scientific report 2025

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