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 1 M glucose solution is effective in eluting CBM9 and CBM9-tagged fusion proteins from a very inexpensive commercially-avail able 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 mg l(-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%. (C) 2004 Elsevier B.V. All rights reserved.