@article {2461,
	title = {Direct Measurement of the Kinetics of CBM9 Fusion-Tag Bioprocessing Using Luminescence Resonance Energy Transfer},
	journal = {Biotechnology Progress},
	volume = {25},
	number = {3},
	year = {2009},
	note = {ISI Document Delivery No.: 462SETimes Cited: 1Cited Reference Count: 44Kavoosi, Mojgan Creagh, A. Louise Turner, Robin F. B. Kilburn, Douglas G. Haynes, Charles A.},
	month = {May-Jun},
	pages = {874-881},
	type = {Article},
	abstract = {The economics of affinity-tagging technologies, particularly at preparative scales, depends in part on the cost and efficiency of the bioprocessing step used to remove the affinity tag and obtain the final purified product (Lowe et al., J Biochem Biophys Methods. 2001,49:561-574). When CBM9, the family 9 cellulose binding module from Thermotoga maritima, serves as the affinity tag, the overall efficiency of tag removal is a function of the choice of processing enzyme and the local structure of the cleavage site, most notably the linker sequence flanking the bioprocessing recognition site on the tag side. A novel spectroscopic method is reported and used to rapidly and accurately measure CBM9 fusion-tag bioprocessing kinetics and their dependence on the choice of linker sequence. The assay monitors energy transfer between a lanthanide-based donor bound to the CBM9 tag and an acceptor fluorophore presented on the tat-get protein or peptide. Enzyme-catalyzed cleavage of the fusion tag terminates this resonance energy transfer, resulting in a change in fluorescence intensity that can be monitored to quantify substrate concentration over time. The assay is simple, fast and accurate, providing k(cat)/K-M values that contain standard errors of less than 3\%. As a result, both substantial and subtle differences in bioprocessing kinetics can be measured and used to guide bioproduct design. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 874-881, 2009},
	keywords = {10A, ACTIVE-SITE, affinity chromatography, BEND ANGLE, CARBOHYDRATE-BINDING MODULE, CONFORMATIONAL-CHANGES, enterokinase, ESCHERICHIA-COLI, fusion tag, green fluorescent protein (GFP), high throughput assay, LANTHANIDE, library, linker, luminescence, protein purification, PURIFICATION, RECOMBINANT PROTEINS, resonance energy transfer (LRET), RNA-POLYMERASE, terbium, THERMOTOGA-MARITIMA, XYLANASE},
	isbn = {8756-7938},
	url = {<Go to ISI>://000267375500030},
	author = {Kavoosi, M. and Creagh, A. L. and Turner, R. F. B. and Kilburn, D. G. and Haynes, C. A.}
}
@article {1225,
	title = {Entropic interaction chromatography: Separating proteins on the basis of size using end-grafted polymer brushes},
	journal = {Biotechnology and Bioengineering},
	volume = {90},
	number = {1},
	year = {2005},
	note = {ISI Document Delivery No.: 909ETTimes Cited: 9Cited Reference Count: 48},
	month = {Apr},
	pages = {1-13},
	type = {Article},
	abstract = {Partitioning of a macromolecule into the interfacial volume occupied by a grafted polymer brush decreases the configurational entropy (Delta S(c)brush) of the terminally attached linear polymer chains due to a loss of free volume. Self-consistent field theory (SCF) calculations are used to show that Delta S(c)brush is a strong function of both the size (MW,) of the partitioning macromolecule and the depth of penetration into the brush volume. We further demonstrate that the strong dependence of Delta S(c)brush on MW, provides a novel and powerful platform, which we call entropic interaction chromatography (EIC), for efficiently separating mixtures of proteins on the basis of size. Two EIC columns, differing primarily in polymer grafting density, were prepared by growing a brush of poly(methoxyethyl acrylamide) chains on the surface of a widepore (1,000-angstrom pores, 64-mu m diameter rigid beads) resin (Toyopearl AF-650M) bearing surface aldehyde groups. Semipreparative 0.1-L columns packed with either EIC resin provide reduced-plate heights of 2 or less for efficient separation of globular protein mixtures over at least three molecular-weight decades. Protein partitioning within these wide-pore EIC columns is shown to be effectively modeled as a thermodynamically controlled process, allowing partition coefficients (K-p) and elution chromatograms to be accurately predicted using a column model that combines SCF calculation of Kp values with an equilibrium-dispersion type model of solute transport through the column. This model is used to explore the dependence of column separation efficiency on brush properties, predicting that optimal separation of proteins over a broad MW, range is achieved at low to moderate grafting densities and intermediate chain lengths. (c) 2005 Wiley Periodicals, Inc.},
	keywords = {ADSORPTION, CHAIN MOLECULES, CONSISTENT-FIELD THEORY, entropic interaction chromatography, equilibrium dispersion, EXCLUSION CHROMATOGRAPHY, GEL FILTRATION, grafted polymer brush, HUMAN SERUM-ALBUMIN, LIQUID-CHROMATOGRAPHY, MODEL, MOLECULAR-WEIGHT, MONTE-CARLO, protein purification, size exclusion chromatography, STATISTICAL-THEORY},
	isbn = {0006-3592},
	url = {<Go to ISI>://000227843800001},
	author = {Pang, P. and Koska, J. and Coad, B. R. and Brooks, D. E. and Haynes, C. A.}
}
@article {847,
	title = {Cyclic enterobacterial common antigen: Potential contaminant of bacterially expressed protein preparations},
	journal = {Journal of Biomolecular Nmr},
	volume = {29},
	number = {2},
	year = {2004},
	note = {ISI Document Delivery No.: 801ZPTimes Cited: 5Cited Reference Count: 10},
	month = {Jun},
	pages = {199-204},
	type = {Article},
	abstract = {We have previously reported the identification of the cyclic enterobacterial common antigen (ECACYC) polysaccharide in E. coli strains commonly used for heterologous protein expression (PJA Erbel et al., J. Bacteriol. 185 ( 2003): 1995). Following this initial report, interactions among several NMR groups established that characteristic N-acetyl signals of ECACYC have been observed in N-15-H-1 HSQC spectra of samples of various bacterially-expressed proteins suggesting that this water-soluble carbohydrate is a common contaminant. We provide NMR spectroscopic tools to recognize ECACYC in protein samples, as well as several methods to remove this contaminant. Early recognition of ECA-based NMR signals will prevent time-consuming analyses of this copurifying carbohydrate.},
	keywords = {enterobacterial common antigen, ESCHERICHIA-COLI, heterologous protein expression, IDENTIFICATION, N-acetylated carbohydrate, NMR-SPECTROSCOPY, protein purification},
	isbn = {0925-2738},
	url = {<Go to ISI>://000220134100007},
	author = {Erbel, P. J. A. and Seidel, R. and Macintosh, S. E. and Gentile, L. N. and Amor, J. C. and Kahn, R. A. and Prestegard, J. H. and McIntosh, L. P. and Gardner, K. H.}
}
