Protein purification by bulk crystallization: The recovery of ovalbumin

Judge R.A., Johns M.R. and White E.T. (1995) Protein purification by bulk crystallization: The recovery of ovalbumin. Biotechnology and Bioengineering, 48 4: 316-323. doi:10.1002/bit.260480404


Author Judge R.A.
Johns M.R.
White E.T.
Title Protein purification by bulk crystallization: The recovery of ovalbumin
Journal name Biotechnology and Bioengineering   Check publisher's open access policy
ISSN 1097-0290
Publication date 1995-01-01
Sub-type Article (original research)
DOI 10.1002/bit.260480404
Open Access Status Not yet assessed
Volume 48
Issue 4
Start page 316
End page 323
Total pages 8
Subject 1305 Biotechnology
1502 Banking, Finance and Investment
2402 Applied Microbiology and Biotechnology
2404 Microbiology
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°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, kGσ 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, kGσ. 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. © 1995 John Wiley & Sons, Inc. Copyright
Keyword bulk crystallization
crystalgrowth rate
nucleation
ovalbumin
purification
Q-Index Code C1
Institutional Status Unknown

Document type: Journal Article
Sub-type: Article (original research)
Collection: Scopus Import - Archived
 
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