Expanded bed chromatography of proteins in small-diameter columns. II. Methods development and scale up.
The scaled down system developed in Part I of this series was further validated by using a 1-cm diameter column for method development studies for the separation of two model proteins, alcohol dehydrogenase and alpha-glucosidase, from unclarified yeast homogenate by hydrophobic interaction expanded bed chromatography based on the STREAMLINE matrix. The efficacy of solids removal and establishment of optimal binding and separation condition by stepwise elution were investigated. Equilibration of the EBA column and loading at high salt strengths affected the subsequent recovery of the two target proteins. Although good resolution between the target proteins could be achieved, peak tailing was found to be a consistent problem. The optimised separation protocol was scaled up 25-fold to a column diameter of 5.0 cm. The results were in good agreement with the run conducted in the 1-cm column, indicating the potential of using the small columns as an viable approach for method scouting and development studies.
Ghose
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School of Engineering & Applied Science, Aston University, Birmingham, UK.
Chase
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D000327:Adsorption; D000426:Alcohol Dehydrogenase / Q000302:isolation & purification*; D002845:Chromatography / Q000295:instrumentation / Q000379:methods*; D011506:Proteins / Q000302:isolation & purification; D012116:Resins, Plant; D013347:Subcellular Fractions / Q000201:enzymology; D015003:Yeasts / Q000201:enzymology; D000520:alpha-Glucosidases / Q000302:isolation & purification*
D011506:Proteins; D012116:Resins, Plant; D000426:Alcohol Dehydrogenase; D000520:alpha-Glucosidases