Site-Specific Modification of Single Domain Antibodies by Enzyme-Immobilized Magnetic Beads

Abstract

Nanobodies as imaging agents and drug conjugates have shown great potential for cancer diagnostics and therapeutics. However, site-specific modification of a nanobody with microbial transglutaminase (mTGase) encounters problems in protein separation and purification. Here, we describe a facile yet reliable strategy of immobilizing mTGase onto magnetic beads for site-specific nanobody modification. The mTGase immobilized on magnetic beads (MB-mTGase) exhibits catalytic activity nearly equivalent to that of the free mTGase, with good reusability and universality. Magnetic separation simplifies the protein purification step and reduces the loss of nanobody bioconjugates more effectively than size exclusion chromatography. Using MB-mTGase, we demonstrate site-specific conjugation of nanobodies with fluorescent dyes and polyethylene glycol molecules, enabling targeted immunofluorescence imaging and improved circulation dynamics and tumor accumulation in vivo. The combined advantages of MB-mTGase method, including high conjugation efficiency, quick purification, less protein loss, and recycling use, are promising for site-specific nanobody functionalization and biomedical applications.

Protein–polymer bioconjugation, immobilization, and encapsulation: a comparative review towards applicability, functionality, activity, and stability

“…22 Soluble protein-polymer bioconjugates can be tuned to produce desired effects for specific biomaterials and have been shown to exhibit a unique array of properties including: improving pharmacokinetic activity, introducing stimuliresponsiveness, and modulating enzymatic activity and stability. 100,101 This section will investigate the advantages and disadvantages of soluble polymer conjugation on industrially, agriculturally, and biomedically relevant proteins.…”

Magnetic Nanoparticle Support with an Ultra-Thin Chitosan Layer Preserves the Catalytic Activity of the Immobilized Glucose Oxidase

“…Like all immobilized enzymes, they allow for higher stability during catalytic reactions and broader temperature and pH stability ranges [ 15 , 16 , 17 , 18 , 19 ]. In addition, magnetic supports grant easy magnetic separation during both the catalyst synthesis and the catalytic reaction as well as magnetic control for continued processes [ 20 , 21 , 22 , 23 , 24 , 25 ]. It is noteworthy that magnetic nanoparticle (NP) morphology is crucial for magnetic properties.…”