Digestion of Flexible Linkers
CD BioGlyco has a professional Glycoengineering Platform with multiple world-leading technologies to help customers comprehensively promote the digestion of flexible linkers.
Designing fusion proteins composed of multiple protein domains is a very popular way of designing new protein functions. The linker not only separates the two protein domains, but its properties directly affect the functional properties of the fusion protein. The most commonly used linkers for linking protein or peptide domains are flexible linkers.
The most commonly used flexible linkers have sequences consisting primarily of stretches of glycine (Gly) and serine (Ser) residues ("GS" linker). Currently, the most widely used flexible linkers have the sequence (Gly-Gly-Gly-Gly-Ser)n. In addition to GS linkers, many other flexible linkers have emerged for recombinant fusion proteins. These flexible linkers, including KESGSVSSEQLAQFRSLD and EGKSSGSGSESKST, have been used to construct biologically active single-chain variable fragments (scFvs). The Gly and Ser residues in the linker were designed to provide flexibility, whereas glutamic acid (Glu) and lysine (Lys) were added to improve the solubility.
Flexible linkers with good flexibility and solubility are often rich in small or polar amino acids, such as Gly and Ser. Flexible linkers with this property are very useful when the fusion protein domains require certain movements or interactions (e.g. scFv).
We utilize a selection of highly specific proteases and engineered enzymes developed in-house to selectively cleave flexible linker regions. These enzymes are optimized for diverse linker sequences, ensuring precise digestion without compromising the structural integrity of the antibody's functional domains.
Intermediate analytical strategies provide a means to rapidly improve mass spectrometry quality. However, the new model brings new analytical challenges.
CD BioGlyco provides specific enzymes to digest flexible glycine-rich fusion protein linkers including GS and polyglycine (G) linkers. This enzyme enables the isolation of different domains of multifunctional fusion proteins to facilitate characterization and increase protein understanding. This facilitates intermediate-level analysis of fusion proteins that can both reduce overall sample complexity and enable domain-specific recognition and monitoring of post-translational modifications. Fusion proteins and various monoclonal antibody fragments containing glycine-rich linkers can be characterized in detail by intermediate methods.
Our expert team will work closely with you to understand your project goals and the unique characteristics of your antibody. We will analyze your specific antibody structure and linker sequence and customize digestion and glycosylation strategies to perfectly meet your specific needs.
This is the core of our professional services. We use specific enzymes to selectively and efficiently digest glycine-rich flexible fusion protein linkers, including GS and polyglycine (G) linkers. This targeted enzymatic digestion technology precisely separates distinct functional domains in multifunctional fusion proteins and monoclonal antibody fragments, providing in-depth insights into antibody structure and function.
Following enzymatic digestion, advanced chromatography techniques are used to remove all impurities, including residual enzyme and linker peptides. Multi-point quality control using advanced mass spectrometry (MS) ensures complete removal of the linker peptide, ensuring product purity and integrity.
The purified fragments are free of steric hindrance, providing an ideal foundation for subsequent glycoengineering. We collaborate with you to perform a variety of customized glycan modifications using enzymatic and chemoenzymatic methods to achieve the desired homogeneous glycoforms.
Upon project completion, we provide a comprehensive report containing a complete breakdown of the experimental steps, quality control data, and analytical results. The final, high-quality glycoengineered product will be delivered to you along with all supporting documentation and a clear follow-up process.
Journal: Scientific Reports
IF: 3.8
Published: 2022
Results: This study investigates the impact of linker sequences on the in vitro and in vivo properties of an anti-vascular endothelial growth factor (VEGF) scFv derived from Bevacizumab. Two linkers were compared: a repetitive GS linker (L2) and a non-repetitive linker (L1) with similar flexibility but lower predicted immunogenicity. In vitro analyses revealed that L1 exhibited superior properties, including a higher monomer ratio, greater thermal stability, and lower immunogenicity scores, though L2 showed approximately threefold higher binding affinity to VEGF. In vivo, both scFvs inhibited angiogenesis more effectively than bevacizumab, while no significant difference in in vivo efficacy was observed between the two linkers. This is the first study to compare scFv linker effects in a zebrafish model, highlighting that non-repetitive linkers offer advantages in developability (stability, monomericity, immunogenicity) without compromising in vivo anti-angiogenic activity.
Fig.1 Binding kinetics of Bevacizumab, L1, and L2 fragments to their antigen, VEGF. (Arslan, et al., 2022)
CD BioGlyco provides one-stop Antibody Digestion Services and ensures the continuity and transparency of the experimental process. We will continue to improve our standards to meet customers' glycobiology research needs with high quality. If you are interested in our services, please contact us for more details without any hesitation.
Reference
