(9f) Novel Thermo-Responsive Fucose Binding Ligands for Glycoprotein Purification By Affinity Precipitation

Carbohydrates are the most abundant organic molecules found in nature, and they are metabolized and synthesized by nearly all organisms.  Recently, focus in understanding the structure and function of glycoproteins has expanded into the rapidly growing field of glycobiology.  However, the complexity and microheterogeneity of these oligosaccharide chains have made identification, isolation, and analysis very difficult.  To aid in this demanding field, sugar binding proteins, or lectins, have emerged as critical tools in glycobiology.   Lectins and lectin-containing biomaterials have been used in separation and identification, such as lectin affinity chromatography.  However, while amenable at bench scale for glycoprotein separation, scale up of this chromatographic process is notably inefficient and expensive.  Column fouling, diffusion limitations, and flow rate constraints are just some of costly disadvantages of this time consuming process.

To address these problems, we designed a recombinant fusion protein that potentially could be used for affinity precipitation purification of glycoproteins without chromatography.  This protein has dual functionality: a fucose binding bacterial lectin from Ralstonia solanacearum (RSL) for carbohydrate affinity, and an elastin-like protein (ELP) thermo-responsive biopolymer for precipitation.  Affinity separation will be achieved by simple inverse temperature cycling (ITC), which consists of temperature oscillation, followed by recovery of the precipitate via filtration or centrifugation.  Separation may also be achieved by fluctuation of the salt concentration, maintaining the integrity of possible target enzymes.  Furthermore, a newly developed extracellular secretion method for high expression level in E. coli is utilized to establish a cost effective process for production and isolation of this protein, achieving a secretion yield of 35 mg/L over a 48 hour cultivation period.

The presentation will detail the design, overexperession and characterization of several lectin-ELP fusions. The successful application of glycoprotein purification using these novel proteins will be demonstrated with a model protein, horseradish peroxidase (HRP), which contains α1,3-fucosylated glycans.  Isolation of this target glycoprotein from a protein mixture in an affinity precipitation process resulted in a pure glycoprotein and a recovery yield of 88%.