Expression And Purification Of O-Glycosidase And Efficacy Research On Enrichment Of Related Cancer Antigens

Glycomics is an emerging subject after genomics and proteomics,which mainly studies the composition structure and function of glycan chains and is widely used in the study of novel biomarkers.Glycosylation is one of the most common post-translational modifications of proteins,mainly divided into N-glycosylation and O-glycosylation.The analysis of Nglycosylation is relatively mature.However,traditional mass spectrometry techniques are not applicable to the study of O-glycosylated protein structural domains because of the diversity of its glycan structure,lack of peptide consensus motifs and the absence of a dedicated template,which brings challenges to the analysis and detection of O-glycan.Deglycosylation is one of the most important tools to study the structure and function of glycoprotein chains,and different types of glycosidases are useful tools for this purpose.Currently,there is a lack of instrumental enzymes for functional studies of O-glycans.Mucins are a class of proteins highly modified by O-glycosylation,and abnormalities in the structure and expression of these mucin glycans are closely related to the development of cancer,and many mucin-like cancer-associated antigens are used as indicators for clinical diagnosis.Therefore,by expressing and purifying O-glycosidase with mucin selectivity,we can provide tool enzymes for O-glycan analysis and detection,and provide potential value for solving barriers to mucin function studies and exploring its relevance to tumors.The aim of this study is to construct mutant plasmids and fusion expression vectors toinactivate and increase protein yield,and then to prepare O-glycosidase solid-phase beads by immobilized enzyme technology as a tool enzyme for enriching mucin,which can be analyzed by glycan mapping to distinguish disease states and provide new ideas to improve the sensitivity of clinical disease detection techniques.Objectives:（1）Purification of the O-glycosidase family by constructing mutant plasmids and fusion expression vectors as an effective tool to study the structure and function of O-glycan chains（2）Preparation of inactivated O-glycosidase solid-phase beads for enrichment of mucin-like cancer-associated antigens and analysis of their specificity for binding to different antigens,including normal and carcinogenic ones.（3）To explore new methods for developing new techniques for clinical disease detection and improve the sensitivity of clinical disease detection techniques based on the O-glycosylation properties of cancer antigens.Methods:（1）Codon optimization of the coding sequences of the O-glycosidase StcE from Escherichia coli and the O-glycoprotein endonuclease（Operator）from Akkermansia muciniphila for recombinant expression in E.coli was performed using Genscipt’s publicly available codon optimization software.Using commercial gene synthesis,the coding sequences of SctE and Operator carrying a 6-histidine（6His）purification tag were loaded into pET28a and pET21a prokaryotic expression vectors to obtain pET28a/6his-StcE and pET21a/6his-Operator expression vectors,respectively.The expression vector pET28a/6his-Sumo-StcE was also constructed by fusing the DNA encoding 6his-Sumo,a pro-soluble tag,with StcE gene DNA using molecular cloning technique.Mutant plasmids pET28a/6his-Sumo-Stc^EE447D and pET21a/6his-Operator^H205A-E206A were obtained by using PCR-based DNA targeted mutagenesis,rendering both inactive for enzymatic activity.（2）The above plasmids were introduced into the Escherichia coli expression strain,and the experimental conditions for efficient expression of the recombinant target protein were optimized by changing the OD600 value of the bacterial broth concentration,the induction temperature,the induction expression time,and the concentration of the inducer IPTG.Mass expression of the target protein using the screened optimal experimental conditions.（3）The target protein was purified by immobilized metallic nickel affinity chromatography using the His tag constructed by the pre-molecular cloning technique.Linear elution and stepwise elution were performed to determine the optimal concentration of imidazole contained in phosphate buffer to improve the purity of the target protein,respectively.（4）The mutation-inactivated target protein was coupled to a hydrogen bromide activated-agarose gel（CNBr-activated Sepharose 4B）,and the binding efficiency of the protein to the gel was optimized by varying the target protein concentration,gel volume,and coupling time to prepare O-glycosidase solid-phase beads.（5）Cells OVCAR-3,BxPC3 and MCF-7 that can secrete the associated mucin MUC16,MUCl and CA19-9 were cultured.The supernatants of semm-free medium from these cells were collected and concentrated using a peristaltic pump concentration device and ultrafiltration centrifuge tubes.The two O-glycosidase solid-phase microspheres prepared above were used to react with the three cell supernatants separately and analyzed by protein immunoblotting for enrichment of relevant cancer antigens.（6）The ability of Stc^EE447D-beads to enrich serum for CA125 was detected using CA125 antibody after the reaction of the inactivated glycosidase Stc^EE447D-beads prepared above with normal human or ovarian cancer patient serum and separation of the legal proteins by SDS-PAGE.Results:（1）Preliminary identification by electrophoresis on agarose gel and identification by outgoing sequencing showed that the mutant plasmid with lost enzyme activity was successfully constructed:pET28a/6his-Sumo-Stc^EE447D和pET21a/6his-Operator^H205A-E206A.（2）Successful introduction of the plasmid into E.coli recipient cells and successful expression of the target protein was determined by observing the negative control without the inducer（IPTG concentration of 0）and the expected molecular weight.（3）Stc^EE447D and Operator^H205A-E206A proteins obtained after purification by metallo-nickel affinity chromatography were detected by SDS-PAGE and Komas Brilliant Blue staining for the isolation and purification of O-glycosidase,both glycosidases were at the expected molecular weight and the purity of the enzymes was estimated to be>90%by optical densitometry.（4）O-glycosidase solid-phase beads Stc^EE447D and Operator^H205A-E206A were successfully prepared,and the coupling efficiencies of 88%and 80%were obtained by the crude quantification of Kaumas Brilliant Blue staining and the determination of BCA protein concentration,which were ready for subsequent experiments.（5）Cell OVCAR-3,MCF7 and BxPC3 serum-free medium supernatants were successfully collected at 600 mL each,concentrated to 30 mL by peristaltic pump concentration equipment,and then continued to be concentrated to 10 mL by centrifugal filter to obtain MUC16,MUC1 and CA19-9 cell supernatant concentrates.The results of incubation with two O-glycosidase solid-phase beads respectively showed that Stc^EE447D could enrich MUC16 and Operator^H205A-E206A could enrich CA19-9;in addition,both O-glycosidase solidphase beads could enrich MUC1.（6）Stc^EE447D-beads showed differences in differentiating serum CA125 levels between normal subjects and ovarian cancer patients,and the trend presented by protein immunoblot analysis correlated with the high and low clinical CA125 assay values.potential application of Oglycosidase solid phase microspheres in studying the diagnostic sensitivity of CA125 test.Conclusion:In this study,two O-glycosidase solid-phase beads were successfully constructed to enrich for mucin-like cancer-related antigens,and MUC16,MUC1 and CA19-9 were successfully enriched.These antigens are clinically relevant to the development of ovarian,breast,lung and pancreatic cancers,and thus these O-glycosidase solid-phase beads are broad-spectmm in clinical detection.Currently,cancer-related antigens are commonly detected in clinical practice by immunofluorescence,radioimmunoassay,enzyme-linked immunoassay and chemiluminescence assay.Among them,the limitations of enzyme-linked immunoassay are that its manual operation is more complicated,time-consuming and the reproducibility of results is poor,while chemiluminescence has advantages in accuracy and reproducibility,but the cost of detection is higher.In this study,based on protein purification and affinity chromatography,immobilized enzyme technology,O-glycosidase solid-phase beads were used as an analytical tool to enrich CA125 antigen in serum of normal subjects and ovarian cancer patients to distinguish normal or disease status,and the results showed that the enrichment results correlated with the high and low clinical detection values of CA125 in ovarian cancer patients.This technique explores new ways to develop new techniques for clinical disease detection and to improve the sensitivity of clinical disease detection techniques based on the O-glycosylation properties of cancer antigens.