Self-activating factor X derivative fused to the C-terminus of a cellulose-binding module: Production and properties
| Autor: | R. Antony J. Warren, M. Marta Guarna, Douglas G. Kilburn, Emily Kwan, Charles A. Haynes, Alisdair B. Boraston, Neil R. Gilkes |
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| Rok vydání: | 2002 |
| Předmět: |
Glycosylation
Stereochemistry Molecular Sequence Data Bioengineering Kidney Cleavage (embryo) Applied Microbiology and Biotechnology chemistry.chemical_compound Bacterial Proteins Species Specificity Cricetinae Enzyme Stability Escherichia coli Animals Cloning Molecular Cellulose Cells Cultured Base Sequence Sepharose C-terminus Substrate (chemistry) Cells Immobilized Enzymes Immobilized Cellulose binding Fusion protein Recombinant Proteins Biochemistry chemistry Factor X Mutagenesis Site-Directed Derivative (chemistry) Protein Binding Biotechnology |
| Zdroj: | Biotechnology and Bioengineering. 79:724-732 |
| ISSN: | 1097-0290 0006-3592 |
| DOI: | 10.1002/bit.10312 |
| Popis: | In this work, a new derivative of FX was engineered. It comprises a cellulose-binding module (CBM) fused to the N-terminus of the truncated light chain (E2FX) of FX and a hexahistidine tag (H6) fused to the C-terminus of the heavy chain. The sequence LTR at the site of cleavage of the activation peptide from the N-terminus of the heavy chain is changed to IEGR to render the derivative self-activating. However, N-linked glycans on the CBM of the derivative blocked its binding to cellulose and those on the activation peptide slowed its activation. Therefore, the sites of N-linked glycosylation on the CBM and on the activation peptide were eliminated by mutation. The final derivative can be produced in good yield by cultured mammalian cells. It is purified easily with Ni2+-agarose, it is self-activating, and it can be immobilized on cellulose. When immobilized on a column of cellulose beads, the activated derivative retains ∼80% of its initial activity after 30 days of continuous hydrolysis of a fusion protein substrate. Under these conditions of operation, the effective substrate:enzyme ratio is >104. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 79: 724–732, 2002. |
| Databáze: | OpenAIRE |
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