Fishing for lectins from diverse sequence libraries by yeast surface display - an exploratory study.
The establishment of a robust technology platform for the expression cloning of carbohydrate-binding proteins remains a key challenge in glycomics. Here we explore the utility of using yeast surface display (YSD) technology in the interaction-based lectin cloning from complete cDNA libraries. This should pave the way for more detailed studies of protein-carbohydrate interactions. To evaluate the performance of this system, lectins representing three different subfamilies (galectins, siglecs, and C-type lectins) were successfully displayed on the surface of Saccharomyces cerevisiae and Pichia pastoris as a-agglutinin and/or alpha-agglutinin fusions. The predicted carbohydrate-binding activity could be detected for three out of five lectins tested (galectin-1, galectin-3, and siaoadhesin). For galectin-4 and E-selectin, no specific carbohydrate-binding activity could be detected. We also demonstrate that proteins with carbohydrate affinity can be specifically isolated from complex metazoan cDNA libraries through multiple rounds of FACS sorting, employing multivalent, fluorescent-labeled polyacrylamide-based glycoconjugates.
Ryckaert
Stefan
S
Department for Molecular Biomedical Research, Unit for Fundamental and Applied Molecular Biology, VIB, Ghent University, Ghent, Belgium.
Callewaert
Nico
N
Jacobs
Pieter P
PP
Dewaele
Sylviane
S
Dewerte
Isabelle
I
Contreras
Roland
R
D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000378:metabolism; D005434:Flow Cytometry; D037483:Galectin 1 / Q000235:genetics / Q000378:metabolism; D037502:Galectin 3 / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D007135:Immunoglobulin Variable Region; D037102:Lectins / Q000235:genetics* / Q000378:metabolism; D008954:Models, Biological; D010843:Pichia / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; D037483:Galectin 1; D037502:Galectin 3; D007135:Immunoglobulin Variable Region; D037102:Lectins