Protein purification by bulk crystallization: The recovery of ovalbumin

Abstract

Crystallization is used industrially for the recovery and purification of many inorganic and organic materials. However, very little is reported on the application of bulk crystallization for proteins. In this work, ovalbumin was selected as a model protein to investigate the feasibility of using bulk crystallization for the recovery and purification of proteins. A stirred 1-L seeded batch crystallizer was used to obtain the crystal growth kinetics of ovalbumin in ammonium sulfate solutions at 30 degrees C. The width of the metastable region, in which crystal growth can occur without any nucleation, is equivalent to a relative supersaturation of about 20. The bulk crystallizations were undertaken within this range (using initial relative supersaturations less than 10) and nucleation was not observed. The ovalbumin concentration in solution was measured by UV absorbance and checked by crystal content measurement. Crystal size distributions were measured both by using a Malvern Mastersizer and by counting crystals through a microscope. The crystal growth rate was found to have a second-order dependence upon the ovalbumin supersaturation. While there is no discernible effect of ammonium sulfate concentration at pH 4.90, there is a slight effect at higher pH values. Overall the effect of ammonium sulfate concentration is small compared to the effect of pH, for which there is a 10-fold increase in the growth rate constant, k(Gsigma) over the range pH 4.6-5.4. To demonstrate the degree of purification which can be achieved by bulk crystallization, ovalbumin was crystallized from a solution containing conalbumin (80,000 Da) and lysozyme (14, 600 Da). After one crystallization and a crystal wash, ovalbumin crystals were produced with a protein purity greater than 99%. No contamination by the other proteins was observed when using overloaded sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) stained with Coomassie blue stain and only trace amounts of lysozyme were observed using a silver stain. The presence of these other proteins in solution did not effect the crystal growth rate constant, k(Gsigma). The study demonstrates the feasibility of using bulk crystallization for the recovery and purification of ovalbumin. It should be readily applicable to other protein systems.

Properties and Characterization of High Capacity Resins for Biochromatography

“…Currently suppliers of biopharmaceutical drugs are looking for inexpensive alternatives to chromatography [5]. However, because of the requirements of high purity and good throughput, these alternatives have not been able to successfully compete with downstream chromatography steps.…”

Evaluation of a model for seeded isothermal batch protein crystallization

“…However, proteins are large macromolecules, thus causing an intrinsic challenge associated with crystallization. As a consequence, protein crystallization is yet to play a well-established role in the bioprocess industries (Jacobsen et al, 1998;Judge et al, 1995Judge et al, , 1998Klyushnichenko, 2003;Saikumar et al, 1998).…”

“…This method is known to yield accurate and reproducible results. For example, when Judge et al (1995) determined ovalbumin crystal growth rates, they compared values calculated from photographs of individual growing crystals to those obtained from crystal size distribution measurements using laser light scattering. They observed excellent agreement between the two methods.…”

Interactions of proteins in aqueous ammonium-sulfate solutions:Mixtures of lysozyme and ovalbumin

“…These two proteins have been studied extensively in connection with the processing of eggs in the foodprocessing industry (Rossi and Schiraldi, 1992) under conditions (e.g., high temperature) that denature proteins. Judge et al (1995) crystallized ovalbumin to a purity of 99% from aqueous ammonium sulfate containing a mixture of ovalbumin, conalbumin and lysozyme. Lysozyme has been selectively precipitated from lysozyme-ovalbumin mixtures at high pH and ammonium sulfate ionic strength (Coen et al, 1997).…”