Prokaryotic Expression, Purification, Activity Identification And Application Of Cysteine Hydrolase IdeS

Antibodies and related products have emerged as one of the most promisingnew drug classes in the biopharmaceutical industry. In the aspects of cancer andautoimmune diseases, it is highlighted increasingly that part of the importance ofantibody drugs, and becomes gradually the concerned focus. Therefore, to ensurethe quality of antibody drugs and to develop new drugs, it is also veryextraordinarily important to research the property and characterization of thedrugs. When macromolecular antibodies are hydrolyzed specifically, it is verycrucial to resolve the structure and function of antibody,ImmunoglobulinG-degrading enzyme of Streptococcus pyogenes (IdeS) is a kind oftypical cysteine hydrolase, cleaving specifically the IgG into smaller fragments inthe hinge area to obtain the complete (Fab)2and Fc moietes. It can be morevisualized and efficient to acquire the structure feature of antibody and get amore detailed analysis of the characteristic.According to the unique selectivity of substrate, it becomes one of theimportant biotechnological tools for the characterization analysis of antibodydrugs. The recent studies showed that the activity of IdeS was higher than the knownhydrolases, like pepsin, papain and Lys-C, avoiding non-specific cleavage that leadto the extra analysis problems. In addition, IdeS allows a fast proteolysis ofmonclonal antibodies, and it expends its application with its stability of theenzyme activity in large scale. Therefore, IdeS is thought to be one of the best digesting IgG tools at present. However, due to the most popular usage of IdeS, therehas not been very suffcicent on the acquisition and preparation of this enzyme atpresent. Seriously, it leads to limiting the scope of its application. As aconsequence, a new strategy has become a hot spot of current reserch as regardsthe recombinant expression of IdeS.Nevertheless, there’s no related reports concerning high-efficent productionof IdeS in domestic yet. Then, to obtain the proteinase IdeS efficiently and verifythe specificity and the activity of IdeS in detail, firstly, we used gene synthesismethod to acquire the active nucleotide sequence section of protease IdeS.Secondly, we added a His6tag by PCR technology in carboxyl nucleotide sequenceof IdeS, being aimed at removing the protease IdeS by purifing when it finishedthe reaction with IgG. Thirdly, we also made use of the prokaryotic expression vectorpGEX-4T-2to acquire a new soluble recombinant protease IdeS efficiently, bymeans of GST-fused protein. The expressed protease IdeS was purified by thereduced glutathione affinity chromatography and nickel metal chelating affinitychromatography, being designed to get the highly active and pure proteinase IdeS.What’s more, we added the enterokinase site between amino terminal and GST, inorder to remove the tag easily. The purified proteinase IdeS was analyzed andidentified with respect to its activity and specificity by common analysis methods,including SDS-PAGE, HPLC-SEC, LC-MS. The results suggested that the proteinaseIdeS has a better specific and higher activity and a relatively stability in different buffer, which could be utilized effectively for protein structure analysis.Activity research showed that the proteinase IdeS exhibited highly activity in thePH range of5.1~8.0, with an optimum at PH6.6and reaction temperature at37℃around.Finally, we adopted a special case analysis of antibody, which was not onlyN-glycosylated in the stationary site of Fc moieties, but also N-glycosylated orO-glycosylated in the sites of Fab moieties. However, the glycosylation can probablycause the immunogenicity reactions in human body. Furthermore, the glycosylationalso plays a role in the plasmatic half-life via binding to neonatal Fcγ receptor(FcγR). Owing to protease IdeS, the antibodies are identified and analyzed exactlyby mass spectrum. Moreover, it provides a good example for the qulity control andverification of antibody drugs in future, and it is dramatically significant as faras research and development of antibodies. This method can also be applied forbiosimilars, biobetters, and next-generation antibodies and Fc-fusion proteins.