Purification of L-asparaginase II by crystallization

Abstract

Here a case study of L-asparaginase II out of a recombinant Escherichia coli is presented. The target protein was obtained by simple cell disintegration and acetone precipitation. The L-asparaginase II has been crystallized in three different forms in the following microbatch crystallization. The rod-shaped crystals (~400 µm edge length) were obtained at either 8°C or 22°C after 17 h by addition of PEG₆₀₀₀. The rectangular-shaped crystals were obtained after further recrystallization of the rod-shaped crystals. The rhombic-shaped crystals formed at 8°C after 12 days when cold ethanol was used instead of PEG₆₀₀₀. All crystallizations were performed in tris-acetate buffer (50 mmol·L^-1, pH 5.1). By crystallization, the specific activity of L-asparaginase II has increased 5-fold. The protein content and the purity of the crystals were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The more concentrated L-asparaginase II out of an extract mixture and the presence of only less minor proteins after crystallization demonstrates that crystallization is an effective and mild method to purify the target protein. The single crystal X-ray diffraction pattern reveals that the crystals are proteins and the X-ray powder diffraction (XRPD) pattern shows clearly that the crystals forming in PEG₆₀₀₀ and ethanol have different crystal structures.

Key words： protein crystallization; L-asparaginase II; purification

Acknowledgments

The authors gratefully acknowledge the friendly support of the project from the team of Prof. Dr. Milton T. Stubbs (MLU, physical biotechnology), especially by his PhD student C. Ursel in the qualifying the protein crystals by single crystal X-ray diffraction.

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