Experienced in custom glycoprotein synthesis, Creative Biolabs has perfected our technical pipelines in glycoprotein remodeling. We would like to share our knowledge and passion in glycoprotein remodeling to facilitate our customers’ research and project development.
Glycosylation can greatly affect intrinsic properties and biological functions of a protein, including folding, stability, immunogenicity, and therapeutic efficacy. Due to the complexity of N-glycosylation processing in the biosynthesis, natural and recombinant glycoproteins often carry heterogeneous N-glycans. One major challenge in dealing with glycoproteins is their heterogeneity. Natural glycoproteins are usually produced as mixtures of glycoforms. It is highly difficult to isolate pure glycoforms by chromatographic techniques. It still a great challenge to prepare homogenous glycoproteins for the functional study of glycoproteins. To address this problem, different chemical and biological strategies have been developed to produce homogeneous glycoproteins, such as total chemical synthesis with chemo-selective ligation, native chemical ligation, chemoenzymatic synthesis, and glycosylation pathway engineering in host expression systems.
Human erythropoietin (EPO) is a glycoprotein hormone, which is composed of a single 165 amino acid residues chain. EPO contains four glycans, three N-glycosylation sites at Asn-24, Asn-38, and Asn-83 and one O-glycosylation site at Ser-126. It is an essential growth factor for the development of red blood cells from bone marrow-derived precursors. EPO has been widely used in the treatment of anemia after chemotherapy.
EPO has been remodeled to carry a homogenous azide-tagged Man3GlcNAc2 glycan, which could be further engineered to carry polysialylated glycan or other functional groups. EPO with a high-mannose type glycan was first treated with Endo-H (ENGase from Streptomyces picatus) to generate the GlcNAc-EPO acceptor with three GlcNAc acceptor sites and then treated with Endo-A (ENGase from Arthrobacter protophormiae) glycosynthase. The transfer reaction and purity of protein was monitored with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis of intact protein.
Fig.1 Reaction scheme for glycan remodeling of EPO. (Yang, 2017)
As a world-leading service provider in custom glycoprotein synthesis, Creative Biolabs employs an efficient chemoenzymatic strategy for glycan remodeling of glycoproteins. A glycosynthase is used to transfer a sugar oxazoline which mimics the transition state of glycan hydrolysis of the GlcNAc residue of protein acceptor. Firstly, deglycosylation of glycoprotein is performed to generate the GlcNAc- or Fucα1,6-GlcNAc-protein acceptor. Then, a desired glycan is transferred by a glycosynthase to reconstitute a homogeneous glycoform. Finally, the glycoprotein with desired functions and properties is achieved.
With years of research and development experience in custom glycoprotein synthesis, Creative Biolabs owns a group of scientists who have distinguished knowledge in glycan remodeling of glycoprotein. We offer turn-key or ala carte services customized to our client’s needs. Please contact us for more information about our glycoprotein remodeling service.