Protein Purification by Ultrafiltration Using a β-Galactosidase Fusion Tag

• Published in: Biotechnology progress Vol. 16; no. 2; pp. 296 - 298
• Main Authors: Sakhamuru, K., Hough, D. W., Chaudhuri, J. B.
• Format: Journal Article
• Language: English
• Published: USA American Chemical Society 2000; American Institute of Chemical Engineers
• Subjects: beta-Galactosidase - chemistry; beta-Galactosidase - genetics; Biological and medical sciences; Biotechnology; Catalyst activity; Chemical Fractionation; Electrophoresis, Polyacrylamide Gel; Enzyme kinetics; Escherichia coli; Escherichia coli - genetics; Factor Xa - genetics; Fractionation; Fundamental and applied biological sciences. Psychology; Glucose 1-Dehydrogenase; glucose dehydrogenase; Glucose Dehydrogenases - genetics; Glucose Dehydrogenases - isolation & purification; Membranes, Artificial; Methods. Procedures. Technologies; Molecular Weight; Others; protein purification; Proteins; Purification; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Sugars; Thermoplasma - enzymology; Thermoplasma acidophilum; Ultrafiltration; Ultrafiltration - methods; Various methods and equipments; Purification; Archaeobacteria; Enzyme; Glucose 1-dehydrogenase; Thermoplasma acidophilum; Protein; Separation method; Ultrafiltration; Glycosidases; Bacteria; Hydrolases; Oxidoreductases; Recombinant protein; Fusion protein; O-Glycosidases; β-Galactosidase
• ISSN: 8756-7938; 1520-6033
• DOI: 10.1021/bp990137x

The use of β‐galactosidase (465 kDa) as a fusion tag for ultrafiltration‐based protein purification has been investigated. The target protein studied was thermophilic glucose dehydrogenase (157 kDa, GDH) from Thermoplasma acidophilum. An expression vector was constructed comprising the lacZ gene fused to a factor Xa cleavage sequence that was attached to the 5′ end of the GDH gene. This gene fusion was expressed in
Escherichia coli JM109 to yield a soluble protein that exhibited activities for both enzymes. Cleavage of this fusion protein (622 kDa) by factor Xa gave two smaller proteins that showed individual β‐galactosidase and GDH activity. A two‐stage diafiltration process for protein purification was used in an ultrafiltration stirred cell. In the first stage, a 500 kDa membrane was used to retain the fusion protein and transmit smaller E. coli host proteins. Approximately 80% of the GDH activity was retained in this step. Following cleavage, the second stage utilized a 300 kDa membrane to fractionate the β‐galactosidase and GDH. No β‐galactosidase was detected in the permeate solutions, and 97% of the GDH activity was recovered in the permeate.