The rapidly increasing applications of monoclonal antibodies (mAbs) in therapy have necessitated the development of mAb production and purification technologies for both academic and industrial usage. Herein, a histidine-tagged cyclic peptide (HHHHHHGSGSGSDC*AWHLGELVWC*T, the disulfide-bonded cysteines of which are indicated by asterisks, named HT25-cyclopeptide) functionalized monolithic material was developed by the metal ion chelation-based approach. The resulting material possessed suitable affinity and peptide ligand density (13.8 mg peptide ligand per mL of material), good porosity (67.1 %), acceptable specific surface area (52.95 m 2 /g), and lots of macropores (4.13 μm). Moreover, excellent antibody-specific selectivity, comparable or even better binding capacity (for dried material, maximum static binding capacity and dynamic binding capacity are about 119.3 mg/g and 17.05 mg/g, respectively) for antibody compared to previously developed affinity materials, acceptable resistance to trypsin digestion, and negligible nonspecific protein adsorption, were also achieved on this novel monolithic material. Compared with the corresponding cyclic peptide-based sepharose material, milder elution conditions were employed for the HT25-cyclopeptide-based monolithic material, which could effectively prevent the aggregation and denaturation of the enriched antibodies. This novel material was then successfully applied to the affinity enrichment and purification of mAbs (including infliximab and rituximab) in different cell culture media or IgG in human serum.
Luo Juan,Yu Yao,Wang Ke,He Sizhe,Wang Longjie,Liang Fangfang,Chin Jason W.,Tang Shan
Catalytic mechanism-based, light-activated traps have recently been developed to identify the substrates of cysteine or serine hydrolases. These traps are hydrolase mutants whose catalytic cysteine or serine are replaced with genetically encoded 2,3-diaminopropionic acid (DAP). DAP-containing hydrolases specifically capture the transient thioester- or ester-linked acyl–enzyme intermediates resulting from the first step of the proteolytic reaction as their stable amide analogs. The trapped substrate fragments allow the downstream identification of hydrolase substrates by mass spectrometry and immunoblotting. In this protocol, we provide a detailed step-by-step guide for substrate capture and identification of the peptidase domain of the large tegument protein deneddylase (UL36 USP ) from human herpesvirus 1, both in mammalian cell lysate and live mammalian cells. Four procedures are included: Procedure 1, DAP-mediated substrate trapping in mammalian cell lysate (~8 d); Procedure 2, DAP-mediated substrate trapping in adherent mammalian cells (~6 d); Procedure 3, DAP-mediated substrate trapping in suspension mammalian cells (~5 d); and Procedure 4, substrate identification and validation (~12–13 d). Basic skills to perform protein expression in bacteria or mammalian cells, affinity enrichment and proteomic analysis are required to implement the protocol. This protocol will be a practical guide for identifying substrates of serine or cysteine hydrolases either in a complex mixture, where genetic manipulation is challenging, or in live cells such as bacteria, yeasts and mammalian cells.
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