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BirA AND mouse

Kim Schilders, Evelien Eenjes, Gabriëla Edel, Anne Boerema de Munck, Marjon Buscop van Kempen, Jeroen Demmers, René Wijnen, Dick Tibboel, Robbert J Rottier
Sox2 is a Sry-box containing family member of related transcription factors sharing homology in their DNA binding domain. Sox2 is important during different stages of development, and previously we showed that Sox2 plays an important role in branching morphogenesis and epithelial cell differentiation in lung development. The transcriptional activity of Sox2 depends on its interaction with other proteins, leading to 'complex-specific' DNA binding and transcriptional regulation. In this study, we generated a mouse model containing a biotinylatable-tag targeted at the translational start site of the endogenous Sox2 gene (bioSox2)...
February 2018: Transgenic Research
Fengchun Zhao, Yuan Tian, Huimin Wang, Jiye Liu, Xiao Han, Zhengyou Yang
Organophosphorus pesticides (OPs) are the most widely used pesticides in agriculture, and OP residues have been broadly reported in food and environmental samples. The aim of this study is to develop a recombinant antibody-based broad-specificity immunoassay for OPs. A phage display library was prepared from a mouse pre-immunized with a generic immunogen of OPs, and a single-chain variable fragment (scFv) antibody was selected. The selected scFv antibody was fused with biotin acceptor domain (BAD) and overexpressed as an inclusion body in Escherichia coli BL21 (DE3)...
September 2016: Analytical and Bioanalytical Chemistry
Huimin Wang, Fengchun Zhao, Xiao Han, Zhengyou Yang
In this article, we reported the development of a biotinylated single-chain variable fragment (scFv) antibody based indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) for parathion-methyl (PM) detection. Firstly, a phage display library was generated using a pre-immunized BALB/C mouse against a specific hapten of PM. After four rounds of panning, the scFv gene fragments were transferred into a secreted expression vector. Then, the scFv antibodies were secreted expressed and screened by IC-ELISA against PM...
October 2016: Protein Expression and Purification
Akhee S Jahan, Maxime Lestra, Lee Kim Swee, Ying Fan, Mart M Lamers, Fikadu G Tafesse, Christopher S Theile, Eric Spooner, Roberto Bruzzone, Hidde L Ploegh, Sumana Sanyal
Posttranslational modifications are central to the spatial and temporal regulation of protein function. Among others, phosphorylation and ubiquitylation are known to regulate proximal T-cell receptor (TCR) signaling. Here we used a systematic and unbiased approach to uncover deubiquitylating enzymes (DUBs) that participate during TCR signaling in primary mouse T lymphocytes. Using a C-terminally modified vinyl methyl ester variant of ubiquitin (HA-Ub-VME), we captured DUBs that are differentially recruited to the cytosol on TCR activation...
February 9, 2016: Proceedings of the National Academy of Sciences of the United States of America
Qin Wang, Ryan T Wagner, Austin J Cooney
Embryonic stem (ES) cells have several unique attributes, the two most important of which are they can differentiate into all cell types in the body and they can proliferate indefinitely. To study the regulation of these phenomena, we developed a regulatable in vivo biotinylation expression system in mouse ES cells. The E. coli biotin ligase gene BirA, whose protein product can biotinylate a 15-aa peptide sequence, called the AviTag, was cloned downstream of an IRES. The primary vector containing the doxycycline controlled transactivator gene tTA and IRES-BirA was knocked into the ROSA26 locus by homologous recombination...
2013: PloS One
Ben C King, Angela D Hamblin, Philip M Savage, Leon R Douglas, Ted H Hansen, Ruth R French, Peter W M Johnson, Martin J Glennie
Attempts to generate robust anti-tumour cytotoxic T lymphocyte (CTL) responses using immunotherapy are frequently thwarted by exhaustion and anergy of CTL recruited to tumour. One strategy to overcome this is to retarget a population of virus-specific CTL to kill tumour cells. Here, we describe a proof-of-principle study using a bispecific conjugate designed to retarget ovalbumin (OVA)-specific CTL to kill tumour cells via CD20. A single-chain trimer (SCT) consisting of MHCI H-2K(b)/SIINFEKL peptide/beta 2 microglobulin/BirA was expressed in bacteria, refolded and chemically conjugated to one (1:1; F2) or two (2:1; F3) anti-hCD20 Fab' fragments...
June 2013: Cancer Immunology, Immunotherapy: CII
Mathia Y Lee, Thomas Lufkin
To circumvent the difficulty of isolating specific cell populations by MACS from dissociated complex animal tissue, when their proportions reached levels similar to that of the background, we developed the "Three-step MACS" strategy. Cells of interest are defined by their expression of a particular gene(s) of interest rather by than their natural cell surface markers or size. A two-component transgenic cell surface protein, for two sequential rounds of MACS, is expressed under the promoter control of the endogenous gene of interest by means of gene targeting and the generation of transgenic tissue...
July 2012: Journal of Biomolecular Techniques: JBT
Benjamin B Bartelle, César A Berríos-Otero, Joe J Rodriguez, Anne E Friedland, Orlando Aristizábal, Daniel H Turnbull
RATIONALE: The formation and maintenance of a functional vasculature is essential for normal embryonic development, and genetic changes that affect the vasculature underlie pathogenesis in many human diseases. In vivo imaging in mouse models is required to understand the full complexity of mammalian vascular formation, which is a dynamic and 3-dimensional process. Optical microscopy of genetically expressed fluorescent reporter proteins offers high resolution but limited depth of penetration in vivo...
March 30, 2012: Circulation Research
Kijung Sung, Michael T Maloney, Jingkun Yang, Chengbiao Wu
In this report, we describe a novel method for producing mature and biologically active mono-biotinylated nerve growth factors (mBtNGF) that can be used for single molecule studies of real-time movement of neurotrophins within axons of neurons. We inserted an AviTag sequence into the C-terminal of the full length mouse preproNGF cDNA and cloned the fusion construct into a pcDNA3.1 mammalian expression vector. We also subcloned the Escherichia coli biotin ligase, BirA, into a pcDNA3.1 vector. These two plasmids were then transiently co-expressed in HEK293FT cells...
September 15, 2011: Journal of Neuroscience Methods
Gaurav Batra, Sheikh Mohd Talha, Satish Kumar Nemani, Nisha Dhar, Sathyamangalam Swaminathan, Navin Khanna
Dengue is a rapidly spreading mosquito-borne viral disease prevalent in over a hundred countries around the world. A definitive identification of dengue infection depends on reliable dengue diagnostic tests. This study describes the design, expression and purification of an in vivo biotinylated chimeric dengue antigen to exploit the high affinity of biotin-streptavidin interaction to detect anti-dengue antibodies. This chimeric antigen incorporates the envelope domain III (EDIII) of the four dengue virus serotypes...
November 2010: Protein Expression and Purification
Yi Liu, Lihui Xu, Xuesi Zeng, Jianfang Sun, Xianhui He
Major histocompatibility complex (MHC) tetramer technology offers a powerful means to study specific T cell populations of interest. To investigate the immune response of H-2Db-restricted CD8+ T cells in immunotherapy, we prepared the H-2Db tetramer and verified its effectiveness in enumerating the CD8+ T cells specific for the lymphocytic choriomeningitis virus (LCMV). First, the cDNA encoding H-2Db heavy chain was cloned by RT-PCR from the spleen of a C57BL/6 mouse. The expression vector for H-2Db-BSP, i...
February 2008: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
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