| 1. |
|
|
| 2. |
- Cicortas Gunnarsson, Lavinia, et al.
(författare)
-
Evolution of a carbohydrate binding module into a protein-specific binder
- 2006
-
Ingår i: Biomolecular Engineering. - : Elsevier BV. - 1389-0344 .- 1878-559X. ; 23:2-3, s. 111-117
-
Tidskriftsartikel (refereegranskat)abstract
- A carbohydrate binding module, CBM4-2, derived front the xylanase (Xyn 10A) of Rhodothermus marinus has been used as a scaffold for molecular diversification. Its binding specificity has been evolved to recognise a quite different target, a human monoclonal IgG4. In order to understand the basis for this drastic change in specificity we have further investigated the target recognition of the IgG4-specific CBMs. Firstly, we defined that the structure target recognised by the selected CBM-variants was the protein and not the carbohydrates attached to the glycoprotein. We also identified key residues involved in the new specificity and/or responsible for the swap in specificity, from xylan to human IgG4. Specific changes present in all these CBMs included mutations not introduced in the design of the library from which the specific clones were selected. Reversion of such mutations led to a complete loss of binding to the target molecule, suggesting that they are critical for the recognition of human IgG4. Together with the mutations introduced at will, they had transformed the CBM scaffold into a protein binder. We have thus shown that the scaffold of CBM4-2 is able to harbour molecular recognition for either carbohydrate or protein structures. (c) 2005 Elsevier B.V. All rights reserved.
|
|
| 3. |
- Cicortas Gunnarsson, Lavinia, et al.
(författare)
-
Molecular engineering of a thermostable carbohydrate-binding module
- 2006
-
Ingår i: Biocatalysis and Biotransformation. - : Informa UK Limited. - 1029-2446 .- 1024-2422. ; 24:1-2, s. 31-37
-
Konferensbidrag (refereegranskat)abstract
- Structure-function studies are frequently practiced on the very diverse group of natural carbohydrate-binding modules in order to understand the target recognition of these proteins. We have taken a step further in the study of carbohydrate-binding modules and created variants with novel binding properties by molecular engineering of one such molecule of known 3D-structure. A combinatorial library was created from the sequence encoding a thermostable carbohydrate-binding module, CBM4-2 from a Rhodothermus marinus xylanase, and phage-display technology was successfully used for selection of variants with specificity towards different carbohydrate polymers (birchwood xylan, Avicel (TM), ivory nut mannan and recently also xyloglucan), as well as towards a glycoprotein (human IgG4). Our work not only generated a number of binders with properties that would suite a range of biotechnological applications, but analysis of selected binders also helped us to identify residues important for their specificities.
|
|
| 4. |
|
|
| 5. |
- Cicortas Gunnarsson, Lavinia
(författare)
-
Novel binding specificities engineered into the scaffold of a carbohydrate binding module
- 2007
-
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.
|
|
| 6. |
- Cicortas Gunnarsson, Lavinia, et al.
(författare)
-
Novel xylan-binding properties of an engineered family 4 carbohydrate-binding module
- 2007
-
Ingår i: Biochemical Journal. - : Portland Press. - 0264-6021 .- 1470-8728. ; 406, s. 209-214
-
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.
|
|
| 7. |
|
|
| 8. |
- Filonova, Lada, et al.
(författare)
-
Synthetic xylan-binding modules for mapping of pulp fibres and wood sections
- 2007
-
Ingår i: BMC Plant Biology. - : BioMed Central. - 1471-2229. ; 7
-
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.
|
|
| 9. |
- Gunnarsson, Lavinia Cicortas, 1977-, et al.
(författare)
-
A carbohydrate binding module as a diversity-carrying scaffold
- 2004
-
Ingår i: Protein Engineering Design & Selection. - : Oxford University Press. - 1741-0126 .- 1741-0134. ; 17:3, s. 213-221
-
Tidskriftsartikel (refereegranskat)abstract
- The growing field of biotechnology is in constant need of binding proteins with novel properties. Not just binding specificities and affinities but also structural stability and productivity are important characteristics for the purpose of large-scale applications. In order to find such molecules, libraries are created by diversifying naturally occurring binding proteins, which in those cases serve as scaffolds. In this study, we investigated the use of a thermostable carbohydrate binding module, CBM4-2, from a xylanase found in Rhodothermus marinus, as a diversity-carrying scaffold. A combinatorial library was created by introducing restricted variation at 12 positions in the carbohydrate binding site of the CBM4-2. Despite the small size of the library (1.6 x 10(6) clones), variants specific towards different carbohydrate polymers (birchwood xylan, Avicel and ivory nut mannan) as well as a glycoprotein (human IgG4) were successfully selected for, using the phage display method. Investigated clones showed a high productivity (on average 69 mg of purified protein/l shake flask culture) when produced in Escherichia coli and they were all stable molecules displaying a high melting transition temperature (75.7 +/- 5.3 degrees C). All our results demonstrate that the CBM4-2 molecule is a suitable scaffold for creating variants useful in different biotechnological applications.
|
|
| 10. |
- Gunnarsson, Lavinia Cicortas, 1977-, et al.
(författare)
-
Engineered xyloglucan specificity in a carbohydrate-binding module
- 2006
-
Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 16:12, s. 1171-1180
-
Tidskriftsartikel (refereegranskat)abstract
- The field of plant cell wall biology is constantly growing and consequently so is the need for more sensitive and specific probes for individual wall components. Xyloglucan is a key polysaccharide widely distributed in the plant kingdom in both structural and storage tissues that exist in both fucosylated and non-fucosylated variants. Presently, the only xyloglucan marker available is the monoclonal antibody CCRC-M1 that is specific to terminal alpha-1,2-linked fucosyl residues on xyloglucan oligo- and polysaccharides. As a viable alternative to searches for natural binding proteins or creation of new monoclonal antibodies, an approach to select xyloglucan-specific binding proteins from a combinatorial library of the carbohydrate-binding module, CBM4-2, from xylanase Xyn10A of Rhodothermus marinus is described. Using phage display technology in combination with a chemoenzymatic method to anchor xyloglucan to solid supports, the selection of xyloglucan-binding modules with no detectable residual wild-type xylan and beta-glucan-binding ability was achieved.
|
|
