Protein purification by ultrafiltration using a beta-galactosidase fusion tag.
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.
Sakhamuru
K
K
Department of Chemical Engineering and Department of Biology & Biochemistry, University of Bath, Bath BA2 7AY, U.K.
Hough
D W
DW
Chaudhuri
J B
JB
D005591:Chemical Fractionation; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015951:Factor Xa / Q000235:genetics; D042843:Glucose 1-Dehydrogenase; D005948:Glucose Dehydrogenases / Q000235:genetics / Q000302:isolation & purification*; D008567:Membranes, Artificial; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification*; D013822:Thermoplasma / Q000201:enzymology; D014462:Ultrafiltration / Q000379:methods*; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics
D008567:Membranes, Artificial; D011993:Recombinant Fusion Proteins; D005948:Glucose Dehydrogenases; D042843:Glucose 1-Dehydrogenase; D001616:beta-Galactosidase; D015951:Factor Xa