Cellulose-binding domain

Figure 3. Multiple sequence alignments of the cellulose binding domain family 1 are presented. These alignments were calculated using the program Qustal (JO),...

Figure 1. The organization of catalytic and non-catalytic domains in cellulases from C. fimi and other bacteria. CfCenA, B and C, and CfCex are the endo- and exo-p- 1, 4-glucanases of C. fimi, ClfX is a translated open reading frame from Cellulomonas flavigena (29), CtEGD and PfEndA are endo-p-1, 4-glucanases from Clostridium thermocellum and Pseudomonas fluorescens, respectively (30,31), The primary structures are drawn approximately to scale and are numbered from the amino terminus of the mature protein ClfX is numbered from the start of the open reading frame. Unshaded areas represent catalytic domains, cross-hatched areas indicate cellulose-binding domains, repeated blocks of amino acids are stippled, and black areas represent linker regions.

The introduction of a glutamic acid (E bold), after each RADI 6 repeat was made to allow cleavage by endoproteinase Glu-C, which cleaves C-terminal to glutamate. A cellulose binding domain (CBD) (Table 1) was selected as the affinity tag, as CBDs bind strongly and specifically to cellulose which is a relatively cheap and abundant purification matrix. The main problem encountered was the low level of peptide recovered. Theoretically, 1 g of fusion protein should give 267mg of peptide, but only 10.1 mg of peptide was recovered after RP-HPLC. 

Francisco JA, Stathopoulos C, Warren RAJ, Kilburn DG, Georgiou G, Specific adhesion and hydrolysis of cellulose by intact Escherichia coli expressing surface anchored cellulase or cellulose binding domains, Biotechnology, 11 491 495, 1993. 

Carrard G., Koivula A., Soderlund H., and Beguin P. 2000. Cellulose-binding domains promote hydrolysis of different sites on crystalline cellulose. Proc. Natl. Acad. Sci., 97,10342-10347. 

Creagh, A. L., Ong, E., Jervis, E., Kilbum, D. G., and Haynes, C. A. 1996. Binding of the cellulose-binding domain of exoglucanase Cex from Cellulomonas fimi to insoluble microcrystalline cellulose is entropically driven. Proc. Natl. Acad. Sci., 93, 12229-12234. 

Gao, P. J., Chen, G. J., Wang, T. H., Zhang, Y. S., and Liu, J. 2001. Non-hydrolytic disruption of crystalline structure of cellulose by cellulose binding domain and linker sequence of cellobiohydrolase I from Penicillium janthinellum. Shengwu Huaxue Yu Shengwu Wuli Xuebao, 33,13-18. 

Kilbum, D. G., Assouline, Z., Din, N., Gilkes, N. R., Ong, E., Tomme, P., and Warren, R. A. J. 1993. Cellulose binding domains properties and applications. Proceedings of the second TRICEL symposium on Trichoderma Reesei cellulases and other hydrolases. Espoo, Finland. In Suominen, R, and Rainikainen, T. (Eds.), Foundation for Biotechnological and Industrial fermentation Research 8,281-290. 

Linder, M., and Teeri, T. T. 1996. The Cellulose-Binding Domain of the Major Cellobiohydrolase of Trichoderma reesei Exhibits True Reversibility and a High Exchange Rate on Crystalline Cellulose. Proc. Natl. Acad. Sci., 93,12251-12255. 

Nagy, T., Simpson, P., Williamson, M. P., Hazlewood, G. P., Gilbert, H. J., and Orosz, L. 1998. All three surface tryptophans in Type Ha cellulose binding domains play a pivotal role in binding both soluble and insoluble ligands. FEBS Lett., 429, 312-316. 

Reinikainen, T. 1994. The Cellulose-Binding Domain of Cellobiohydrolase I from Trichoderma reesei (pg. 206). Espoo, Finland VTT publications. 

Lemos, M. A., Teixeira, J. A., Domingues, M. R. M., Mota, M., and Gama, F. M., The enhancement of the cellulolytic activity of cellobiohydrolase I and endoglucanase by the addition of cellulose binding domains derived from Trichoderma reesei. Enzyme Microbial Technol 2003, 32, (1), 35—40. 

Levy, I., and Shoseyov, O., Cellulose-binding domains Biotechnological applications. Biotechnol Advances 2002, 20 (3-4), 191-213. 

An efficient system for the production of recombinant antibodies is cellulose-assisted refolding technology, as described by Berdichevsky et al. [7]. The expressed scFvs were fused to a cellulose-binding domain (CBD) from the bacterium Clostridium thermocellum in the format scFv-CBD. The resulting fusion proteins were obtained in high yield from bacterially produced inclusion bodies that become solubilized and then refold while immobilized on cellulose. The refolded and purified scFv-CBD fusion proteins can be used to form cellulose-based affinity matrices or, as described herein, can be immobilized on a cellulose matrix that makes up part of the immunoelectrochemical sensor device. 

Berdichevsky, Y. Ben-Zeev, E. Lamed, R. Benhar, I., Phage display of a cellulose binding domain from Clostridium thermocellum and its application as a tool for antibody engineering,. /. Immunol. Methods 1999, 228, 151-162... 

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