One-step Immobilization-purification Of CBD-EndoS For Antibodies Fc-glycan Remodeling

Endoglycosidase S(EndoS,EC 3.2.1.96)is an endo-?-N-acetylglucosaminidase found in Streptococcus pyogenes,which can specifically hydrolyze the N-glycans on Fc domain of human IgG antibodies.In recent years,two new glycosynthase mutants(EndoS D233 A and D233Q)were generated from EndoS by site-directed mutagenesis and demonstrated to have transglycosylation activity capable of transferring predefined complex type N-glycan oxazoline to the Fc-deglycosylated IgG antibodies.Accordingly,a novel chemoenzymatic strategy,by application of EndoS and the glycosynthase mutants in combination in vitro,or along with other glycotransferase,has been developed as an efficient approach to generate a variety of antibodies with homogeneous glycoforms for function and therapeutic studies,such as antibody-dependent cellular cytotoxicity(ADCC)and complement-dependent cytotoxicity(CDC).Thus,EndoS and the glycosynthase mutants are highly useful biocatalysts with potential applications in biopharmaceutical industry.However,there are still some key problems to be solved in the industrial application of EndoS and its glycosyltransferase mutants.Firstly,EndoS enzymes were recombinant expressed in Escherichia coli and the production remained at low level which caused high cost.In addition,free enzymes are frequently used as biocatalysts for chemoenzymatic remodeling N-glycan of IgG,which limit their practical applications in industry field because of the tedious downstream purification process and the difficulty in recovery and recycling.Therefore,developing EndoS immobilization technology will be an effective approach to overcome the aforementioned disadvantages and improve the value in industrial application.In this work,a one-step immobilization-purification strategy was developed to immobilize EndoS and its mutants,which were successfully applied in antibody Fc N-glycan remodeling in vitro.At first,the endoglycosidase(EndoS and its glycosynthase mutants D233 A,D233Q)gene was fused with cellulose binding domain(CBD)using pET-35 b vector and transferred into E.Coli.After that,the fusion protein CBD-EndoS and mutants were noncovalently immobilized onto cellulose with high efficiency over 81% by one-step immobilizationpurification.Moreover,the cellulose immobilized CBD-EndoS and mutants presented high catalytic activity and were used for chemoenzymatic Fc glycan remodeling of a therapeutic antibody.Finally,operational and storage stability of immobilized CBD-EndoS and mutants were subsequently assayed.The immobilized CBD-EndoS and mutants could retain over 80% activity after 5 batches and could retain over 85% activity after 30 days of storage at 4 ?.