Protein Purification by Ultrafiltration Using a β‐Galactosidase Fusion Tag

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.