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Yu P.,Jiangsu Simcere Pharmaceutical R and D | Yu P.,Jiangsu Key Laboratory of Molecular Targeted Antitumor Drug Research | Qin G.,Jiangsu Simcere Pharmaceutical R and D | Qin G.,Jiangsu Key Laboratory of Molecular Targeted Antitumor Drug Research | And 3 more authors.
Analytical Biochemistry

A liquid chromatographic method was developed to determine the modification degree of PEGylated proteins. This method effectively separated free polyethylene glycol (PEG) from other species in conjugation mixtures on a C4 reversed-phase column using water-acetonitrile gradient elution. Then the concentrations of free PEG were determined according to the integrated area under the curve of its evaporative light scattering detector (ELSD) signal, which was normalized by the PEG standard with similar molecular weights. The actual numbers of PEG attached to proteins, not those of lysines modified, were calculated. This method was performed with PEGylated arginase mixtures as an example and showed clear advantages over 2,4,6-trinitrobenzenesulfonic acid (TNBS) assays. © 2009 Elsevier Inc. All rights reserved. Source

Wang S.,China Pharmaceutical University | Wang S.,Jiangsu Key Laboratory of Molecular Targeted Antitumor Drug Research | Lv J.,China Pharmaceutical University | Lv J.,Jiangsu Key Laboratory of Molecular Targeted Antitumor Drug Research | And 7 more authors.
Cancer Immunology, Immunotherapy

Co-stimulatory molecules can be efficiently produced as a recombinant protein in E. coli with a large range of applications in the fields of immunotherapy. However, whether, different fusions that would affect their functions have rarely been analyzed. To explore the effects of different fusions and linkers on the molecular conformation and activity of CD137 ligand (CD137L), a recombinant human CD137L protein (rhCD137L) library, which consists of the entire extracellular domain of human CD137L fused to N- or C-terminal His-tag through different linkers, was constructed and all rhCD137Ls were, respectively, expressed in E. coli BL21 (DE3) strain carrying a chaperone plasmid pG-Tf2. After purification of the soluble rhCD137Ls, the recombinant fusion proteins could markedly promote the growth of activated T cells, but their effects on cytokine productions were different from each other. The present work indicated that, although all rhCD137Ls have desired biological activity, different fusions and linkers did affect their structures and functions. Consequently, rational design of a library is a necessary and feasible approach for fusion proteins in order to obtain a satisfactory drug candidate for further development. © Springer-Verlag 2011. Source

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