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- Cicortas Gunnarsson, Lavinia
(författare)
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Novel binding specificities engineered into the scaffold of a carbohydrate binding module
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The growing field of biotechnology is in constant need of proteins that can function as recognition tools for separational, analytical and therapeutic purposes. Different molecular engineering approaches are applied on natural proteins in order to create variants with desired properties. This thesis is based on five original papers that deal with selection, characterisation and application of novel binding specificities engineered into the scaffold of a carbohydrate binding module that originates from a xylanase in the thermophilic bacterium Rhodothermus marinus. Molecular evolution studies on this scaffold allowed for the generation of variants that bind specifically to the carbohydrate targets xylan, Avicel?, mannan and xyloglucan. In addition, the scaffold employed in this work was also able to adopt specific protein recognition to a human IgG4 molecule. Apart from high binding specificities, the engineered proteins have additional properties such as high thermal stability and ease of production in Escherichia coli, which are advantageous in most applications. One of the papers in this thesis demonstrates the potential use of the created xylan-binding variants for detection of their target in wood fibres and plant sections. Also, generated variants with other binding specificities have the potential to find similar use as bioanalytical tools. In conclusion, the scaffold of the carbohydrate binding module engaged in the engineering studies of this thesis proved to be suitable for carrying diversity and has thus allowed for the creation of novel variants with diverse binding specificities useful in biotechnological applications.
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| 2. |
- Cicortas Gunnarsson, Lavinia, et al.
(författare)
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Novel xylan-binding properties of an engineered family 4 carbohydrate-binding module
- 2007
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Ingår i: Biochemical Journal. - : Portland Press. - 0264-6021 .- 1470-8728. ; 406, s. 209-214
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Tidskriftsartikel (refereegranskat)abstract
- Molecular engineering of ligand-binding proteins is commonly used for identification of variants that display novel specificities. Using this approach to introduce novel specificities into CBMs (carbohydrate-binding modules) has not been extensively explored. Here, we report the engineering of a CBM, CBM42 from the Rhodothermits marinus xylanase Xyn10A, and the identification of the X-2 variant. As compared with the wildtype protein, this engineered module displays higher specificity for the polysaccharide xylan, and a lower preference for binding xylo-oligomers rather than binding the natural decorated polysaccharide. The mode of binding of X-2 differs from other xylan-specific CBMs in that it only has one aromatic residue in the binding site that can make hydrophobic interactions with the sugar rings of the ligand. The evolution of CBM4-2 has thus generated a xylan-binding module with different binding properties to those displayed by CBMs available in Nature.
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| 3. |
- Filonova, Lada, et al.
(författare)
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Synthetic xylan-binding modules for mapping of pulp fibres and wood sections
- 2007
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Ingår i: BMC Plant Biology. - : BioMed Central. - 1471-2229. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The complex carbohydrate composition of natural and refined plant material is not known in detail but a matter that is of both basic and applied importance. Qualitative assessment of complex samples like plant and wood tissues requires the availability of a range of specific probes. Monoclonal antibodies and naturally existing carbohydrate binding modules (CBMs) have been used in the past to assess the presence of certain carbohydrates in plant tissues. However, the number of natural CBMs is limited and development of carbohydrate-specific antibodies is not always straightforward. We envisage the use of sets of very similar proteins specific for defined targets, like those developed by molecular evolution of a single CBM scaffold, as a suitable strategy to assess carbohydrate composition. An advantage of using synthetic CBMs lies in the possibility to study fine details of carbohydrate composition within non-uniform substrates like plant cell walls as made possible through minor differences in CBM specificity of the variety of binders that can be developed by genetic engineering.RESULTS: A panel of synthetic xylan-binding CBMs, previously selected from a molecular library based on the scaffold of CBM4-2 from xylanase Xyn10A of Rhodothermus marinus, was used in this study. The wild type CBM4-2 and evolved modules both showed binding to wood sections. However, differences were observed in the staining patterns suggesting that these modules have different xylan-binding properties. Also the staining stability varied between the CBMs, the most stable staining being obtained with one (X-2) of the synthetic modules. Treatment of wood materials resulted in altered signal intensities, thereby also demonstrating the potential application of engineered CBMs as analytical tools for quality assessment of diverse plant material processes.CONCLUSION: In this study we have demonstrated the usefulness of synthetic xylan-binding modules as specific probes in analysis of hemicelluloses (xylan) in wood and fibre materials.
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