Intracellular protein crystallization in living insect cells

Area of Science:

• Structural biology
• Biophysics
• X-ray crystallography

Background:

• Crystallization of recombinant proteins in living cells is an emerging technique.
• This method offers a quasi-native environment, avoiding protein purification.
• Existing methods require systematic screening strategies for intracellular crystal growth.

Purpose of the Study:

• To present a detailed protocol for the InCellCryst pipeline.
• To optimize intracellular microcrystal production for serial X-ray diffraction.
• To utilize cellular compartments as screening parameters for crystal growth.

Main Methods:

• Optimized target gene expression using a baculovirus vector system.
• Intracellular crystal formation and detection within living insect cells.
• Serial X-ray diffraction data collection directly from cells.

Main Results:

• Successful production of homogeneous microcrystals within living insect cells.
• Demonstration of serial X-ray diffraction experiments using these intracellular crystals.
• Potential for diffraction data collection within 24 days of target gene cloning.

Conclusions:

• The InCellCryst pipeline provides a systematic and versatile strategy for intracellular protein crystallization.
• This approach facilitates rapid structural determination using X-ray diffraction.
• Intracellular crystallization is a powerful complement to conventional protein crystallization techniques.