Shanghai Medicilon Inc.
Shanghai Medicilon Inc.
Fan L.-J.,China Pharmaceutical University |
Fan L.-J.,Shanghai Medicilon Inc. |
Lv L.-L.,Shanghai Medicilon Inc. |
Liu Y.,China Pharmaceutical University |
Chen C.-L.,Shanghai Medicilon Inc.
Chinese Journal of Pharmaceutical Biotechnology | Year: 2014
To obtain the active antigen-binding fragment (Fab) of Adalimumab from E. coli by constructing a secretory expression system using "double gene" expression vector, the cDNA of light chain and heavy chain of Adalimumab fab along with signal peptide PelB and OmpA respectively, were cloned into vector pETDuet-1. The resulting plasmids was transformed in BL21 (DE3) Star, induced by IPTG at low temperature. The active Adalimumab Fab protein was obtained through the periplasmic extraction, affinity purification, ion exchange and size exclusion chromatography. The Adalimumab Fab protein was analyzed by SDS-PAGE, Western blot and ELISA. SDS-PAGE and Western Blot analysis showed that both heavy chain and light chain were successfully translocated into the periplasm of E. coli and the formed heterologous dimer. The purified Adalimumab Fab protein from multipul purification steps could interact with its antigen TNF-α. Active Adalimumab Fab protein was successfully obtained from E. coli periplasm using "double gene" expression vector, which provided an economic and convenient method for the study on biological function and industry production of Adalimumab Fab. © 2014, Editorial Board of Pharmaceutical Biotechnology. All right reserved.
Tang S.-R.,China Pharmaceutical University |
Wang T.,Shanghai Medicilon Inc. |
Wang M.,China Pharmaceutical University
Chinese Journal of Pharmaceutical Biotechnology | Year: 2015
The objective of this study was to obtain the active compound protein of ecdysteroid receptor (EcR) and ultraspiracle (USP) by constructing a co-expression system which was using expression vectors of EcR and USP to co-express efficiently in E. coli. The target gene of EcR along with N-His tag was cloned into vector pET21bMDX12(+) to build the plasmid pET21bMDX12(+)-EcR, and the target gene of USP along with N-His tag was cloned into vector pET28a(+) to build the plasmid pET28a(+)-USP. Both of EcR and USP plasmids were co-transformed in expression host strain BL21(DE3), then after, IPTG was added to induce coexpression at low temperature. The active EcR-USP compound protein was extracted by the most common method ultrasonication, and purified through affinity purification (Ni-NTA affinity column) and size exclusion chromatography (HiLoad16/60 Superdex 200 molecular sieve). The EcR-USP compound protein was analyzed by identification methods such as SDS-PAGE, Western Blot and MicroCal iTC200 to identify the purity and activity of the target protein. The result of SDS-PAGE and Western Blot analysis showed that both EcR and USP were successfully translocated into expression host strain BL21 (DE3) and formed heterologous dimer. The result of MicroCal iTC200 showed that the purified EcR-USP compound protein from multipul purification steps could interact with ecdysone 20E so that it could prove that the EcR-USP compound protein was active. The conclusion of this study was that the active and soluble EcR-USP compound protein of bombyx mori was successfully obtained from E. coli, which provided an economic and convenient method for the study on biological function and industrial and agricultural production of EcR-USP. At the same time, the success of the co-expression system of EcR-USP in E. coli laid the foundation for the further study of the structure and function of EcR-USP. This study also provided a favorable condition to the crystal structure research of EcR-USP in the future. © 2015, Editorial Board of Pharmaceutical Biotechnology. All right reserved.
Yin Z.,China Pharmaceutical University |
Yin Z.,University of Wollongong |
Song Y.,Shanghai University |
Rehse P.H.,Shanghai Medicilon Inc.
ACS Chemical Biology | Year: 2013
Phosphorylation-dependent protein-protein interaction has rarely been targeted in medicinal chemistry. Thymoquinone, a naturally occurring antitumor agent, disrupts prephosphorylated substrate recognition by the polo-box domain of polo-like kinase 1, a key mitotic regulator responsible for various carcinogenesis when overexpressed. Here, crystallographic studies reveal that the phosphoserine/phosphothreonine recognition site of the polo-box domain is the binding pocket for thymoquinone and its analogue poloxime. Both small molecules displace phosphopeptides bound with the polo-box domain in a slow but noncovalent binding mode. A conserved water bridge and a cation-π interaction were found as their competition strategy against the phosphate group. This mechanism sheds light on small-molecule intervention of phospho-recognition by the polo-box domain of polo-like kinase 1 and other phospho-binding proteins in general. © 2012 American Chemical Society.
Garrison K.,Earle A Chiles Research Institute |
Hahn T.,Earle A Chiles Research Institute |
Lee W.-C.,Shanghai Medicilon Inc. |
Ling L.E.,Biogen Idec |
And 2 more authors.
Cancer Immunology, Immunotherapy | Year: 2012
Effective tumor immunotherapy may require not only activation of anti-tumor effector cells, but also abrogation of tumor-mediated immunosuppression. The cytokine TGF-β, is frequently elevated in the tumor microenvironment and is a potent immunosuppressive agent and promoter of tumor metastasis. OX40 (CD134) is a member of the TNF-α receptor superfamily and ligation by agonistic antibody (anti-OX40) enhances effector function, expansion, and survival of activated T cells. In this study, we examined the therapeutic efficacy and antitumor immune response induced by the combination of a small molecule TGF-β signaling inhibitor, SM16, plus anti-OX40 in the poorly immunogenic, highly metastatic, TGF-β-secreting 4T1 mammary tumor model. Our data show that SM16 and anti-OX40 mutually enhanced each other to elicit a potent anti-tumor effect against established primary tumors, with a 79% reduction in tumor size, a 95% reduction in the number of metastatic lung nodules, and a cure rate of 38%. This positive treatment outcome was associated with a 3.2-fold increase of tumor-infiltrating, activated CD8+ T cells, an overall accumulation of CD4+and CD8+ T cells, and an increased tumor-specific effector T cell response. Complete abrogation of the therapeutic effect in vivo following depletion of CD4+ and CD8+ T cells suggests that the anti-tumor efficacy of SM16+ anti-OX40 therapy is T cell dependent. Mice that were cured of their tumors were able to reject tumor re-challenge and manifested a significant tumor-specific peripheral memory IFN-γ response. Taken together, these data suggest that combining a TGF-β signaling inhibitor with anti-OX40 is a viable approach for treating metastatic breast cancer. © Springer-Verlag 2011.
Zhu J.,Fudan University |
Zhu J.,Shanghai Medicilon Inc. |
Yang Q.,Fudan University |
Dai D.,Fudan University |
Huang Q.,Fudan University
Journal of the American Chemical Society | Year: 2013
To better understand the structural origins of inhibitor selectivity of human phosphodieasterase families (PDEs 1-11), here we report the X-ray crystal structure of PDE2 in complex with a highly selective, nanomolar inhibitor (BAY60-7550) at 1.9 Å resolution, and the structure of apo PDE2 at 2.0 Å resolution. The crystal structures reveal that the inhibitor binds to the PDE2 active site by using not only the conserved glutamine-switch mechanism for substrate binding, but also a binding-induced, hydrophobic pocket that was not reported previously. In silico affinity profiling by molecular docking indicates that the inhibitor binding to this pocket contributes significantly to the binding affinity and thereby improves the inhibitor selectivity for PDE2. Our results highlight a structure-based design strategy that exploits the potential binding-induced pockets to achieve higher selectivity in the PDE inhibitor development. © 2013 American Chemical Society.
Zhou Y.,China Pharmaceutical University |
Jianhua C.,China Pharmaceutical University |
Rehse P.H.,Shanghai Medicilon Inc.
Journal of Medical Colleges of PLA | Year: 2010
Objective: To provide a kinetic model(s) and reveal the mechanism of thymoquinone and Poloxin blocking an emerging anti-cancer target, human Polo-like kinase 1 (hPlk1) Polo-box domain (PBD). Methods: The binding kinetics was determined by using a fluorescence polarization based assay. The putative mechanism was examined with a competition test. Results: Thymoquinone follows a one-step binding with an association rate constant (k1) of 6.635×103 L·mol-1·min-1. Poloxin fit a two-step binding with a dissociation constant (Ki) of 118 μmol/L for the intermediate complex and its isomerization rate (k4) of 0.131 5 min-1 to form an irreversible adduct. No significant dissociation was observed for either ligand up to 13 h. The inhibitors responded insignificantly to the presence of Michael donors as hPlk1-PBD competitors. Conclusion: Thymoquinone and Poloxin are slow-tight ligands to the hPlk1-PBD with kinetic models distinct from each other. Michael addition as the mechanism is excluded. © 2010 The Editorial Board of Journal of Medical Colleges of PLA.
Zhang C.,Merck And Co. |
Ondeyka J.,Merck And Co. |
Herath K.,Merck And Co. |
Jayasuriya H.,Merck And Co. |
And 9 more authors.
Journal of Natural Products | Year: 2011
Platensimycin (1a) and platencin (2) are inhibitors of FabF and FabF/H bacterial fatty acid synthase. The discovery of natural congeners is an approach that can render a better understanding of the structure-function relationships of complex natural products. The isolation and structure elucidation of nine new congeners (11-20) of platensimycin and platencin are described from a fermentation broth of Streptomyces platensis. These hydroxylated congeners are likely derived by cytochrome P450 oxidation of the terpenoid units post-cyclization. Polar groups in the terpenoid portion of the molecule produce negative interactions with the hydrophobic pocket of FabF, resulting in poor activities. However, the discovery of these compounds serves an important purpose, not only to understand structure-function relationships, which cannot be easily accessed by chemical modification, but also to provide access to compounds that could be used for structural identification/confirmation of the oxidative trace metabolites produced in vivo during animal experiments. © 2011 The American Chemical Society and American Society of Pharmacognosy.