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Zhang Z.,Beijing Institute of Biotechnology | Zhang Z.,Beijing Institute of Microbiology and Epidemiology | Liu M.,Beijing Institute of Biotechnology | Liu M.,East China University of Science and Technology | And 6 more authors.
Journal of Biomolecular Structure and Dynamics | Year: 2013

The SNP -158G>A of KLK3 has been validated as a regulatory SNP (rSNP) by molecular biology assays, but the mechanism of how it affects the binding of an androgen receptor (AR) homodimer with DNA is unclear. In the current study, molecular dynamics simulation was adopted to explain its inner cause. Based on a recent review), three types of intermolecular forces were analyzed, and the differences among them were compared between complexes containing -158 A:T and -158 G:C. Extra hydrophobic contacts caused by the methyl group on the mutated thymine were the most crucial factor to the regulatory effect of this rSNP. Further analysis concerning the relative motion of the two recognition helixes of the AR homodimer indicated that the hydrophobic interactions between the recognition helix B and the major groove containing -158 A:T changed that helix's motion greatly from swaying in a plane at free state to vibrating slightly around an equilibrium position. A relatively full explanation on the occurrence of rSNP -158G>A is presented here. Copyright © 2012 Taylor & Francis.

Coxon A.,Amgen | Bready J.,Amgen | Min H.,Amgen | Min H.,Samsung | And 35 more authors.
Molecular Cancer Therapeutics | Year: 2010

AMG 386 is an investigational first-in-class peptide-Fc fusion protein (peptibody) that inhibits angiogenesis by preventing the interaction of angiopoietin-1 (Ang1) and Ang2 with their receptor, Tie2. Although the therapeutic value of blocking Ang2 has been shown in several models of tumorigenesis and angiogenesis, the potential benefit of Ang1 antagonism is less clear. To investigate the consequences of Ang1 neutralization, we have developed potent and selective peptibodies that inhibit the interaction between Ang1 and its receptor, Tie2. Although selective Ang1 antagonism has no independent effect in models of angiogenesis-associated diseases (cancer and diabetic retinopathy), it induces ovarian atrophy in normal juvenile rats and inhibits ovarian follicular angiogenesis in a hormone-induced ovulation model. Surprisingly, the activity of Ang1 inhibitors seems to be unmasked in some disease models when combined with Ang2 inhibitors, even in the context of concurrent vascular endothelial growth factor inhibition. Dual inhibition of Ang1 and Ang2 using AMG 386 or a combination of Ang1- and Ang2-selective peptibodies cooperatively suppresses tumor xenograft growth and ovarian follicular angiogenesis; however, Ang1 inhibition fails to augment the suppressive effect of Ang2 inhibition on tumor endothelial cell proliferation, corneal angiogenesis, and oxygen-induced retinal angiogenesis. In no case was Ang1 inhibition shown to (a) confer superior activity to Ang2 inhibition or dual Ang1/2 inhibition or (b) antagonize the efficacy of Ang2 inhibition. These results imply that Ang1 plays a context-dependent role in promoting postnatal angiogenesis and that dual Ang1/2 inhibition is superior to selective Ang2 inhibition for suppression of angiogenesis in some postnatal settings. ©2010 AACR.

Liu M.,Beijing Institute of Biotechnology | Liu M.,East China University of Science and Technology | Liu M.,Genor Biopharma Co. | Wang S.,Beijing Institute of Biotechnology | And 5 more authors.
PLoS ONE | Year: 2012

Amylosucrase (AS) is a kind of glucosyltransferases (E.C. belonging to the Glycoside Hydrolase (GH) Family 13. In the presence of an activator polymer, in vitro, AS is able to catalyze the synthesis of an amylose-like polysaccharide composed of only α-1,4-linkages using sucrose as the only energy source. Unlike AS, other enzymes responsible for the synthesis of such amylose-like polymers require the addition of expensive nucleotide-activated sugars. These properties make AS an interesting enzyme for industrial applications. In this work, the structures and topology of the two AS were thoroughly investigated for the sake of explaining the reason why Deinococcus geothermalis amylosucrase (DgAS) is more stable than Neisseria polysaccharea amylosucrase (NpAS). Based on our results, there are two main factors that contribute to the superior thermostability of DgAS. On the one hand, DgAS holds some good structural features that may make positive contributions to the thermostability. On the other hand, the contacts among residues of DgAS are thought to be topologically more compact than those of NpAS. Furthermore, the dynamics and unfolding properties of the two AS were also explored by the gauss network model (GNM) and the anisotropic network model (ANM). According to the results of GNM and ANM, we have found that the two AS could exhibit a shear-like motion, which is probably associated with their functions. What is more, with the discovery of the unfolding pathway of the two AS, we can focus on the weak regions, and hence designing more appropriate mutations for the sake of thermostability engineering. Taking the results on structure, dynamics and unfolding properties of the two AS into consideration, we have predicted some novel mutants whose thermostability is possibly elevated, and hopefully these discoveries can be used as guides for our future work on rational design. © 2012 Liu et al.

Xue J.-H.,Genor Biopharma Co. | Huang X.-Y.,Genor Biopharma Co. | Xu S.-Q.,Genor Biopharma Co.
Chinese Journal of Biologicals | Year: 2015

Objective: To develop a real-time quantitative PCR method for determination of copy number of light chain and heavy chain genes of foreign antibody in transgenic CHO cells. Methods: The standard curves of light and heavy chain genes were generated with the standard plasmids containing light and heavy chain genes respectively. The genomic DNA of CHO cells transfected with light chain and heavy chain genes were extracted and analyzed by real-time quantitative PCR. According to the number of single copy gene in 10 ng CHO genomic DNA, the copy number of light chain and heavy chain genes were calculated by the standard curve. Results: The standard curves for copy numbers of light and heavy chain genes were generated respectively, of which the correlation coefficients were more than 0. 99. The amplification efficiencies of light and heavy chain genes were 91. 6% and 91. 8% respectively, indicating good specificities of the curves. The copy numbers of light chain and heavy chain genes decreased simultaneously with the increasing passage of cultured cells. Conclusion: A real-time quantitative PCR method for determination of copy numbers of light and heavy chain genes was developed successfully, which might be used for the study on genetic stability of foreign antibody genes in CHO cells, and provided a test method for obtaining the cell lines for high expression.

Zhang G.,Genor Biopharma Co. | Zhang H.,Genor Biopharma Co. | Nian W.,Genor Biopharma Co. | Sun J.,Genor Biopharma Co.
Shengwu Gongcheng Xuebao/Chinese Journal of Biotechnology | Year: 2011

We established a stable Chinese hamster ovary (CHO-S) cell line for recombinant human VEGF 165-expressing. We co-transfected GS-expression vector and rhVEGF 165 expression plasmid into CHO-S cells, and selected the highest VEGF 165-expressing clone as the working cell line to express VEGF 165 protein. After 7-day fed-batch culture in a 5 L bioreactor and 3 steps chromatographic purification, we got the rhVEGF 165 protein for series of binding and biological activity examination. The production was over 50 mg/L. The purified rhVEGF 165 protein was functionally active with a half-maximal Human Umbilical Vein Endothelial Cells (HUVEC) growth-enhancing effect concentration of 1.94 ng/mL. It was slightly better than commercially available Escherichia coli expressing rhVEGF 165. So we expressed successfully rhVEGF 165 protein in high-level and obtained the fully active rhVEGF 165 protein in large quantity. © 2011 CJB, All rights reserved.

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