Inexpensive and generic affinity purification of recombinant proteins using a family 2a CBM fusion tag.
The selective binding of the family 2a carbohydrate binding module (CBM2a) of xylanase 10A of the soil bacterium Cellulomonas fimi to a variety of cellulosic substrates is shown to provide a new, cost-effective affinity chromatography system for purification of recombinant protein. Genetic linkage of CBM2a to a target protein, in this case protein A from Staphylococcus aureus, results in a fusion protein that binds strongly to the particulate-cellulose resin Avicel PH101 and retains the biological activity of the fusion partner. Affinity purification of protein A-CBM2a from the supernatant of a recombinant E. coli JM101 culture results in a product purity of greater than 95% and a product concentration factor of 34 +/- 3. Measured column parameters are combined with one-dimensional equations governing continuity and intraparticle diffusion to predict product breakthrough curves with good accuracy over the range of realistic operating conditions. Peak spreading within the column is controlled by intraparticle diffusion for CBM2a and by a combination of film mass transfer and intraparticle diffusion for the larger protein A-CBM2a fusion protein.
The Biotechnology Laboratory and the Protein Engineering Network of Centres of Excellence, The University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3.
Beatriz
B
Rodriguez
Mojgan
M
Kavoosi
Jürgen
J
Koska
A Louise
AL
Creagh
Douglas G
DG
Kilburn
Charles A
CA
Haynes
D002170:Canada; D040143:Cellulomonas / Q000201:enzymology*; D002846:Chromatography, Affinity / Q000191:economics / Q000379:methods*; D003198:Computer Simulation; D004926:Escherichia coli / Q000378:metabolism*; D037102:Lectins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D008956:Models, Chemical; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D013205:Staphylococcal Protein A / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D037102:Lectins; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A