An expression system for screening of proteins for glycan and protein interactions

作者：Otto Diana M E; Campanero Rhodes Maria A; Karamanska Rositsa; Powell Andrew K; Bovin Nicolai; Turnbull Jeremy E; Field Robert A; Blackburn Jonathan; Feizi Ten; Crocker Paul R^*

来源：Analytical Biochemistry, 2011, 411(2): 261-270.

DOI：10.1016/j.ab.2010.12.036

• Cloning
• Expression
• Lectin
• Glycan
• Protein

Here we describe a versatile high-throughput expression system that permits genome-wide screening of type 1 membrane and secreted proteins for interactions with glycans and proteins using both cell-expressed and soluble forms of the expressed proteins. Based on Gateway cloning methodology, we have engineered a destination vector that directs expression of enhanced green fluorescent protein (EGFP)-tagged proteins at the cell surface via a glycosylphosphatidylinositol tail. The EGFP fusion proteins can then be cleaved with PreScission protease to release soluble forms of proteins that can be optionally biotinylated. We demonstrate the utility of this cloning and expression system for selected low-affinity membrane lectins from the siglec family of sialic acid-binding immunoglobulin-like lectins, for the glycosaminoglycan-binding proteins FGF-1 and BACE, and for the heterotypic adhesion molecules JAM-B and JAM-C. Cell-expressed proteins can be evaluated for glycan interactions using polyvalent soluble glycan probes and for protein interactions using either cells or soluble proteins. Following cleavage from the cell surface, proteins were complexed in solution and sufficient avidity was achieved to measure weak protein-glycan and weak protein-protein interactions using glycan arrays and surface plasmon resonance, respectively.

• 出版日期2011-4-15