The use of beta-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 beta-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 beta-galactosidase and GDH. No beta-galactosidase was detected in the permeate solutions, and 97% of the GDH activity was recovered in the permeate.
|Number of pages||3|
|Publication status||Published - 2000|