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Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

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Cleaning protocols for crystallization robots: preventing protease contamination.

Andreas Naschberger¹, Barbara G Fürnrohr¹, Theresia Dunzendorfer-Matt¹

¹Division of Biological Chemistry (Biocenter), Medical University of Innsbruck, Innrain 80, 6020 Innsbruck, Austria.

Acta Crystallographica. Section F, Structural Biology Communications

|January 24, 2015

Summary

This summary is machine-generated.

Commercial enzyme cleaners like Zymit contain proteases resistant to EDTA inhibition. Effective removal requires strong alkaline conditions (0.1 M NaOH) to prevent protein degradation during sensitive procedures.

Keywords:

Zymit cleaning protocol crystallization robotics protease protease inhibitor

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Area of Science:

Biochemistry
Protein Chemistry
Enzyme Kinetics

Background:

Commercial enzyme cleaners are widely used for protein removal.
Zymit is a common low-foam enzyme cleaner.
Metalloprotease inhibitors like EDTA are standard for blocking enzyme activity.

Purpose of the Study:

To evaluate the effectiveness of EDTA and other agents in inhibiting Zymit protease activity.
To determine optimal conditions for complete deactivation of Zymit protease.
To prevent protein degradation in sensitive experimental setups.

Main Methods:

Testing Zymit protease inhibition with Ethylenediaminetetraacetic acid (EDTA) at various concentrations.
Assessing protein degradation in crystallization drops after different wash steps.
Evaluating the efficacy of Sodium Dodecyl Sulfate (SDS) and Sodium Hydroxide (NaOH) washes.

Main Results:

EDTA up to 5 mM did not completely inhibit Zymit protease.
Significant protein degradation occurred with EDTA-containing washes.
0.1% SDS washes were insufficient to remove residual Zymit activity.
At least 0.1 M NaOH was required to completely deactivate Zymit protease.

Conclusions:

Zymit protease activity is not fully inhibited by standard EDTA concentrations.
Strong alkaline washes (≥0.1 M NaOH) are essential for complete Zymit protease deactivation.
Adherence to protocols like ZENM involving strong NaOH washes is recommended for sensitive protein experiments.