Apply 20 mg of Herceptin onto the column, and wash with 15 mL of the washing buffer. and the subsequent enzymatic reglycosylation using a glycosynthase mutant such as Endo-S D233A to afford a homogeneous glycoform of the antibody [9C11]. More recently, novel glycosynthase mutants were generated from Endo-S2 that exhibited much enhanced catalytic efficiency over the Endo-S enzyme. In combination with the use Pronase E of an -fucosidase, non-fucosylated glycoforms of antibodies could be generated, that showed much higher ADCC than the initial antibodies [12, 13]. We have previously explained the general protocols for the chemoenzymatic glycoengineering of intact antibodies to produce homogeneous antibody glycoforms [14, 15]. In this paper, we describe a detailed protocol of the chemoenzymatic defucosylation of Herceptin (i.e., trastuzumab, a mAb drug widely used for the treatment of breast malignancy) as a model system with the focus on the comparison of defucosylation by two bacterial -fucosidases. One is the -fucosidase AlfC from that Rabbit polyclonal to ISYNA1 shows both hydrolysis and transglycosylation activity [16C18]. The other is the -fucosidase BfFuc from that demonstrates very broad substrate specificity [12,13]. The schematic procedures are shown in Fig. 1. The protocol includes (1) Fc deglycosylation using the endoglycosidase S2 (Endo-S2); (2) enzymatic defucosylation of the producing Fuca1,6GlcNAc-Herceptin by two unique bacterial -fucosidases, the AlfC and BfFuc; (3) reglycosylation of the GlcNAc-Herceptin using an Endo-S2 glycosynthase mutant (Endo-S2 D184M) as the enzyme and a complex N-glycan oxazo-line as the donor substrate; and (4) SPR analysis of the binding of antibody glycoforms with the FclllA receptor. The comparative studies showed that this -fucosidase AlfC was much more efficient for defucosylation of the Fc than the -fucosidase BfFuc and the non-fucosylated Fc glycoform exhibited significantly higher affinity for the FclllA receptor than that of the commercial Herceptin. Open in a separate windows Fig. 1 Chemoenzymatic glycan remodeling of monoclonal antibody Herceptin. The procedures include enzymatic defucosylation of the therapeutic antibody by a combined use of endoglycosidase S2 and an -fucosidase, followed by enzymatic transfer of an N-glycan using glycosynthase Endo-S2 D184M to afford a non-fucosylated homogeneous glycoform of Herceptin 2.?Materials All chemicals, reagents, and solvents were purchased from Sig-ma-Aldrich and TCI and used as received unless specially noted. The -fucosidases AlfC from BL23 (GenBankaccession no. “type”:”entrez-protein”,”attrs”:”text”:”CAQ67984.1″,”term_id”:”190713978″,”term_text”:”CAQ67984.1″CAQ67984.1) and BfFuc from NCTC 9343 (GenBank accession no. “type”:”entrez-protein”,”attrs”:”text”:”CAH08937.1″,”term_id”:”60494145″,”term_text”:”CAH08937.1″CAH08937.1) were cloned and overexpressed as previously described [13,18,19]. The recombinant proteins Endo-S2 WT and Endo-S2 D184M were prepared according to the procedures explained previously . The sugar oxazolines used in this protocol were prepared using previously reported methods . 2.1. Antibody, Enzymes, and Substrates Recombinant monoclonal antibody (mAb) Herceptin? (tras-tuzumab) (Genentech, Inc.) (Clean five moments with 4 mL of PBS buffer, and resuspend the antibody into PBS buffer (pH 7.4, 10 g/L, final). Purification of Herceptin using Proteins A column: Proteins A affinity chromatography was utilized to purify Herceptin. Clean the column with 5 mL of Proteins A column elution buffer, and equilibrate the column with 10 mL of Proteins A column cleaning buffer at a flowrate of just one 1 mL/min. 20 mg of Herceptin onto Pronase E the column Apply, and clean with 15 mL from the cleaning buffer. Add 100 L of Proteins A column neutralization buffer into small fraction pipes (1 mL for every) for the collector. The bound Herceptin with 20 mL from the elution buffer Elute. Pool the fractions including Herceptin. Transfer the mixed fractions to a centrifuge filtration system device (10 kDa MWCO), and perform the buffer exchange as referred to above. LC-MS evaluation of antibody: Add 1 L of Herceptin (1 g/L) into 19 L of ddH2O including 0.1% formic acidity (was inserted into pET22b-CPD vector, Pronase E and enzyme was overexpressed in BL21 (DE3) like a fusion proteins having a C-terminal cysteine protease site (CPD) carrying a 10 x His label at 20.