Recombinant L‐Asparaginase B and its Crystallization – What is the Nature of Protein Crystals?

Abstract

L-Asparaginase is a protein produced by Escherichia coli and exists in two types, A and B. Only type B shows a specific antitumor activity to acute lymphatic leukemia. A new recombinant E. coli BL21Gold (DE3) pET11a-ansB was used to produce L-asparaginase B in higher quantities. To enhance the purity and preserve the activity of L-asparaginase B, crystallization is a promising process. Based on available patents, first experiments on L-asparaginase B crystallization were performed. Recombinant L-asparaginase B was successfully and reproducibly crystallized in two modifications. Up to now, it is not clear in which respect these two modifications of crystalline L-asparaginase B differ. It is part of the current study to investigate the nature of protein crystals in detail. The investigations on lysozyme crystals are well in agreement with the literature.

Purification of Recombinant L‐Asparaginase II Using Solvent‐Freeze‐Out Technology

“…E. coli BL21Gold (DE3) (kindly provided by the group of Prof. Markus Pietzsch), was used as the bacterial host for protein expression. The E. coli cells were transformed with the plasmid pET11a‐ansB as described in the literature . The whole cultivation followed the procedures described by Müller .…”

“…The E. coli cells were transformed with the plasmid pET11a‐ansB as described in the literature . The whole cultivation followed the procedures described by Müller . After 24 hours of cultivation, the biomass was harvested using centrifugation, washed with a 0.9 wt % NaCl solution, and stored at −80 °C.…”

Determination of the phase diagram for the crystallization of L‐asparaginase II by a turbidity technique

“…L‐asparaginase II was extracted from frozen recombinant Escherichia coli cells which were cultivated according to the description of Müller et al with the following modifications: the cells were grown in a fed‐batch with an initial culture volume of 11.5 L medium and with a start concentration of 20 g/L glucose at 37 °C. After 8.5 h the enzyme production was induced by addition of 1 mM IPTG.…”

Determination of the phase diagram for the crystallization of L‐asparaginase II by a turbidity technique ‐ part II. ‐MPD and crystallography studies‐

“…The activity capacity of L‐asparaginase II can be estimated by calculating the amount of released ammonia from the absorbance at 436nm according to the description of Müller et al. . The activity value is expressed in the enzyme unit, U, where 1U demonstrates the amount of enzyme that catalyzes the formation of 1 μmol of ammonia at 37 °C in 1 minute .…”