Inexpensive one-step purification of polypeptides expressed in Escherichia coli as fusions with the family 9 carbohydrate-binding module of xylanase 10A from T. maritima.
A novel inexpensive affinity purification technology is described based on recombinant expression in Escherichia coli of the polypeptide or protein target fused through its N-terminus to TmXyn10ACBM9-2 (CBM9), the C-terminal family 9 carbohydrate-binding module of xylanase 10A from Thermotoga maritima. Measured association constants (K(a)) for adsorption of CBM9 to insoluble allomorphs of cellulose are between 2 x 10(5) and 8 x 10(6) M(-1). CBM9 also binds a range of soluble sugars, including glucose. As a result, a 1M glucose solution is effective in eluting CBM9 and CBM9-tagged fusion proteins from a very inexpensive commercially-available cellulose-based capture column. A processing site is encoded at the C-terminus of the tag to facilitate its rapid and quantitative removal by Factor X(a) to recover the desired target protein sequence following affinity purification. Fusion of the CBM9 affinity tag to the N-terminus of green fluorescent protein (GFP) from the jellyfish, Aquorin victoria, is shown to yield >200 mgl(-1) of expressed soluble fusion protein that can be affinity separated from clarified cell lysate to a purity of >95% at a yield of 86%.
Kavoosi
Mojgan
M
The Department of Chemical and Biological Engineering, The University of British Columbia, Vancouver, BC, Canada V6T 1Z3.
Meijer
Julia
J
Kwan
Emily
E
Creagh
A Louise
AL
Kilburn
Douglas G
DG
Haynes
Charles A
CA
D001483:Base Sequence; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D020124:Thermotoga maritima / Q000201:enzymology*; D014995:Xylosidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D014995:Xylosidases