TY - JOUR
T1 - Affinity maturation generates greatly improved xyloglucan-specific carbohydrate binding modules
AU - Von Schantz, Laura
AU - Gullfot, Fredrika
AU - Scheer, Sebastian
AU - Filonova, Lada
AU - Cicortas Gunnarsson, Lavinia
AU - Flint, James E.
AU - Daniel, Geoffrey
AU - Nordberg-Karlsson, Eva
AU - Brumer, Harry
AU - Ohlin, Mats
N1 - Funding Information:
These studies were supported by grants from the Swedish Research Council to MO and from Formas to HB. HB is a Fellow (Rådforskare) of the Swedish Research Council. LF and GD were financed by the Formas Funcfiber Centre and Wallenbergstiftelsen. We thank Farid Ibatullin (KTH) for preparing xylogluco-oligosaccharides and Dr. Qi Zhou (KTH) for supplying biotinylated xyloglucans. The CCRC-M1 antibody was made available to us in part supported by NSF grant RCN-0090281. HB and FG thank Prof. Harry Gilbert for his hospitality, scientific insight, and access to ITC equipment during a brief study visit to Newcastle.
PY - 2009/10/31
Y1 - 2009/10/31
N2 - Background: Molecular evolution of carbohydrate binding modules (CBM) is a new approach for the generation of glycan-specific molecular probes. To date, the possibility of performing affinity maturation on CBM has not been investigated. In this study we show that binding characteristics such as affinity can be improved for CBM generated from the CBM4-2 scaffold by using random mutagenesis in combination with phage display technology. Results: Two modified proteins with greatly improved affinity for xyloglucan, a key polysaccharide abundant in the plant kingdom crucial for providing plant support, were generated. Both improved modules differ from other existing xyloglucan probes by binding to galactose-decorated subunits of xyloglucan. The usefulness of the evolved binders was verified by staining of plant sections, where they performed better than the xyloglucan-binding module from which they had been derived. They discriminated non-fucosylated from fucosylated xyloglucan as shown by their ability to stain only the endosperm, rich in non-fucosylated xyloglucan, but not the integument rich in fucosylated xyloglucan, on tamarind seed sections. Conclusion: We conclude that affinity maturation of CBM selected from molecular libraries based on the CBM4-2 scaffold is possible and has the potential to generate new analytical tools for detection of plant carbohydrates.
AB - Background: Molecular evolution of carbohydrate binding modules (CBM) is a new approach for the generation of glycan-specific molecular probes. To date, the possibility of performing affinity maturation on CBM has not been investigated. In this study we show that binding characteristics such as affinity can be improved for CBM generated from the CBM4-2 scaffold by using random mutagenesis in combination with phage display technology. Results: Two modified proteins with greatly improved affinity for xyloglucan, a key polysaccharide abundant in the plant kingdom crucial for providing plant support, were generated. Both improved modules differ from other existing xyloglucan probes by binding to galactose-decorated subunits of xyloglucan. The usefulness of the evolved binders was verified by staining of plant sections, where they performed better than the xyloglucan-binding module from which they had been derived. They discriminated non-fucosylated from fucosylated xyloglucan as shown by their ability to stain only the endosperm, rich in non-fucosylated xyloglucan, but not the integument rich in fucosylated xyloglucan, on tamarind seed sections. Conclusion: We conclude that affinity maturation of CBM selected from molecular libraries based on the CBM4-2 scaffold is possible and has the potential to generate new analytical tools for detection of plant carbohydrates.
UR - http://www.scopus.com/inward/record.url?scp=71049184894&partnerID=8YFLogxK
U2 - 10.1186/1472-6750-9-92
DO - 10.1186/1472-6750-9-92
M3 - Article
C2 - 19878581
AN - SCOPUS:71049184894
SN - 1472-6750
VL - 9
JO - BMC Biotechnology
JF - BMC Biotechnology
M1 - 92
ER -