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Protein expression and purification

Peggy Stolt-Bergner, Christian Benda, Tim Bergbrede, Hüseyin Besir, Patrick H N Celie, Cindy Chang, David Drechsel, Ariane Fischer, Arie Geerlof, Barbara Giabbai, Joop van den Heuvel, Georg Huber, Wolfgang Knecht, Anita Lehner, Regis Lemaitre, Kristina Nordén, Gwynn Pardee, Ines Racke, Kim Remans, Astrid Sander, Judith Scholz, Magda Stadnik, Paola Storici, Daniel Weinbruch, Isabel Zaror, Linda H L Lua, Sabine Suppmann
Baculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production. Most laboratories have preference in using either the E...
March 12, 2018: Journal of Structural Biology
V Vasilca, A Sadeghpour, S Rawson, L E Hawke, S A Baldwin, T Wilkinson, D Bannister, V L G Postis, M Rappolt, S P Muench, L J C Jeuken
Screening assays performed against membrane protein targets (e.g. phage display) are hampered by issues arising from protein expression and purification, protein stability in detergent solutions and epitope concealment by detergent micelles. Here, we have studied a fast and simple method to improve screening against membrane proteins: spherical-supported bilayer lipid membranes ("SSBLM"). SSBLMs can be quickly isolated via low-speed centrifugation and redispersed in liquid solutions while presenting the target protein in a native-like lipid environment...
March 12, 2018: Analytical Biochemistry
Alborz Karimzadeh, Vanessa M Scarfone, Erika Varady, Connie Chao, Karin Grathwohl, John W Fathman, David A Fruman, Thomas Serwold, Matthew A Inlay
Hematopoietic stem cells (HSCs) are the self-renewing multipotent progenitors to all blood cell types. Identification and isolation of HSCs for study has depended on the expression of combinations of surface markers on HSCs that reliably distinguish them from other cell types. However, the increasing number of markers required to isolate HSCs has made it tedious, expensive, and difficult for newcomers, suggesting the need for a simpler panel of HSC markers. We previously showed that phenotypic HSCs could be separated based on expression of CD11a and that only the CD11a negative fraction contained true HSCs...
March 15, 2018: Stem Cells Translational Medicine
Sureyya Mert Selimoglu, Murat Kasap, Gurler Akpinar, Aynur Karadenizli, Abdul Mounem Wis, Uzay Gormus
Human creatine kinase MB (hCKMB) is one of the most preferred biomarkers used for the diagnosis of acute coronary syndrome due to its high sensitivity and specificity. The increasing need for highly purified and biologically active hCKMB in the field of diagnostics makes its production valuable. Currently, the production of hCKMB is mainly achieved in methylotrophic yeast, Pichia pastoris, because the production in Escherichia coli is challenging and generally yields an inactive enzyme with a low quantity. With the aim of finding the best way for the high-yield production of active hCKMB in E...
March 14, 2018: Journal of Molecular Microbiology and Biotechnology
Dionysios C Watson, Bryant C Yung, Cristina Bergamaschi, Bhabadeb Chowdhury, Jenifer Bear, Dimitris Stellas, Aizea Morales-Kastresana, Jennifer C Jones, Barbara K Felber, Xiaoyuan Chen, George N Pavlakis
The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the establishment of reproducible, scalable, and high-throughput methods for the production and purification of clinical grade EV. Methods including ultracentrifugation (U/C), ultrafiltration, immunoprecipitation, and size-exclusion chromatography (SEC) have been employed to isolate EV, each facing limitations such as efficiency, particle purity, lengthy processing time, and/or sample volume. We developed a cGMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving bioreactor culture, tangential flow filtration (TFF), and preparative SEC...
2018: Journal of Extracellular Vesicles
Imran Khan, Patricia S Steeg
The NM23/NME gene was identified as a metastasis suppressor. It's re-expression inhibited cancer cell motility and suppressed metastasis, without effecting primary tumor size in multiple model systems. The mechanisms of NME suppression of motility and metastasis are incompletely known. Of particular interest, has been NME histidine 118 phosphorylation, involved in nucleoside diphosphate kinase (NDPK) and histidine protein kinase (HPK) activities. Using recently developed monoclonal antibodies to phosphohistidine, we have addressed the correlation of NME phosphohistidine with motility suppression, and distinguished the NDPK and HPK contributions...
February 13, 2018: Oncotarget
Chady Nasrallah, Karine Rottier, Romain Marcellin, Vincent Compan, Joan Font, Amadeu Llebaria, Jean-Philippe Pin, Jean-Louis Banères, Guillaume Lebon
The metabotropic glutamate (mGlu) receptors are class C G protein-coupled receptors (GPCRs) that modulate synaptic activity and plasticity throughout the mammalian brain. Signal transduction is initiated by glutamate binding to the venus flytrap domains (VFT), which initiates a conformational change that is transmitted to the conserved heptahelical domains (7TM) and results ultimately in the activation of intracellular G proteins. While both mGlu1 and mGlu5 activate Gαq G-proteins, they also increase intracellular cAMP concentration through an unknown mechanism...
March 13, 2018: Scientific Reports
Tomoyuki Hatano, Salvatore Alioto, Emanuele Roscioli, Saravanan Palani, Scott T Clarke, Anton Kamnev, Juan Ramon Hernandez-Fernaud, Lavanya Sivashanmugam, Bernardo Chapa-Y-Lazo, Alexandra M E Jones, Robert C Robinson, Karuna Sampath, Masanori Mishima, Andrew D McAinsh, Bruce L Goode, Mohan K Balasubramanian
Actins are major eukaryotic cytoskeletal proteins, which perform many important cell functions, including cell division, cell polarity, wound healing, and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively presently for biochemical studies of actin cytoskeleton from other organisms / cell types. Here we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Pichia pastoris Actin is expressed as a fusion with the actin-binding protein thymosin β4 and purified using an affinity tag introduced in the fusion...
March 13, 2018: Journal of Cell Science
Jun Jiang, Shulin Tang, Jianhong Xia, Jikai Wen, Shuang Chen, Xiaodong Shu, Michael S Y Huen, Yiqun Deng
Wnt/β-catenin signaling activity is maintained in homeostasis by an expanding list of molecular determinants. However, the molecular components and the regulatory mechanisms involved in its fine-tuning remain to be determined. Here, we identified C9orf140, a tumor-specific protein, as a novel Axin1-interacting protein by tandem-affinity purification and mass spectrometry. We further showed that C9orf140 is a negative regulator of Wnt/β-catenin signaling in cultured cells as well as in zebrafish embryos. It functions upstream of β-catenin, outcompetes PP2A for binding to Axin1, influences the balance between phosphorylation and de-phosphorylation of β-catenin, and ultimately compromises Wnt3A-induced β-catenin accumulation...
March 13, 2018: Oncogene
Jeffrey W Habig, Aaron Rowland, Matthew G Pence, Cathy X Zhong
Resistance genes (R-genes) from wild potato species confer protection against disease and can be introduced into cultivated potato varieties using breeding or biotechnology. The R-gene, Rpi-vnt1, which encodes the VNT1 protein, protects against late blight, caused by Phytophthora infestans. Heterologous expression and purification of active VNT1 in quantities sufficient for regulatory biosafety studies was problematic, making it impractical to generate hazard characterization data. As a case study for R-proteins, a weight-of-evidence, tiered approach was used to evaluate the safety of VNT1...
March 9, 2018: Regulatory Toxicology and Pharmacology: RTP
Søren Heissel, Jakob Bunkenborg, Max Per Kristiansen, Anne Fich Holmbjerg, Marie Grimstrup, Ejvind Mørtz, Thomas Kofoed, Peter Højrup
Recombinantly expressed biopharmaceutical proteins often undergo a series of purification steps with the aim of removing contaminating material. Depending on the application of the protein, there are various requirements for the degree of purity, but host cell proteins (HCPs) will in general remain in small amounts. LC-MS has emerged as an orthogonal technique, capable of providing detailed information regarding the individual proteins. The aim of this case study was to characterize the HCPs associated with a biopharmaceutical protein, provided by Statens Serum Institut (DK), which is used in the field of tuberculosis and has not previously been studied by LC-MS...
March 8, 2018: Protein Expression and Purification
Benjamin Pyles, Barbara J Bailus, Henriette O'Geen, David J Segal
The ability to activate or repress specific genes in the brain could have a tremendous impact for understanding and treating neurological disorders. Artificial transcription factors based on zinc finger, TALE, and CRISPR/Cas9 programmable DNA-binding platforms have been widely used to regulate the expression of specific genes in cultured cells, but their delivery into the brain represents a critical challenge to apply such tools in live animals. In previous work, we developed a purified, zinc finger-based artificial transcription factor that could be injected systemically, cross the blood-brain barrier, and alter expression of a specific gene in the brain of an adult mouse model of Angelman syndrome...
2018: Methods in Molecular Biology
Caifeng Xiong, Jie Yuan, Zhiying Wang, Siyao Wang, Chenchen Yuan, Lili Wang
In this work, 2-methacryloyloxyethyl phosphorylcholine (MPC) was used as a ligand to prepare a novel mixed-mode chromatography (MMC) stationary phase by the thiol-ene click reaction onto silica (MPC-silica). It was found that this MPC-silica showed the retention characteristics of hydrophilic interaction chromatography (HILIC) and weak cation exchange chromatography (WCX) under suitable mobile phase conditions. In detail, acidic and basic hydrophilic compounds and puerarin from pueraria were separated quickly with HILIC mode...
March 6, 2018: Journal of Chromatography. A
Alejandro Saettone, Jyoti Garg, Jean-Philippe Lambert, Syed Nabeel-Shah, Marcelo Ponce, Alyson Burtch, Cristina Thuppu Mudalige, Anne-Claude Gingras, Ronald E Pearlman, Jeffrey Fillingham
BACKGROUND: The chromatin remodelers of the SWI/SNF family are critical transcriptional regulators. Recognition of lysine acetylation through a bromodomain (BRD) component is key to SWI/SNF function; in most eukaryotes, this function is attributed to SNF2/Brg1. RESULTS: Using affinity purification coupled to mass spectrometry (AP-MS) we identified members of a SWI/SNF complex (SWI/SNFTt ) in Tetrahymena thermophila. SWI/SNFTt is composed of 11 proteins, Snf5Tt , Swi1Tt , Swi3Tt , Snf12Tt , Brg1Tt , two proteins with potential chromatin-interacting domains and four proteins without orthologs to SWI/SNF proteins in yeast or mammals...
March 9, 2018: Epigenetics & Chromatin
Laura L Burger, Charlotte Vanacker, Chayarndorn Phumsatitpong, Elizabeth R Wagenmaker, Luhong Wang, David P Olson, Suzanne M Moenter
GnRH neurons are a nexus of fertility regulation. We utilized translating ribosome affinity purification coupled with RNA sequencing to examine mRNAs of GnRH neurons in adult intact and gonadectomized (GDX) male and female mice. GnRH neuron ribosomes were tagged with green fluorescent protein (GFP) and GFP-labeled polysomes isolated by immunoprecipitation, producing one RNA fraction enhanced for GnRH neuron transcripts and one RNA fraction depleted. cDNA libraries were created from each fraction and 50-base, paired-end sequencing done and differential expression (enhanced fraction/depleted fraction) determined with a threshold of >1...
March 7, 2018: Endocrinology
Kai Feng, Zhi-Sheng Xu, Jie-Xia Liu, Jing-Wen Li, Feng Wang, Ai-Sheng Xiong
This study showed that a galactosyltransferase, AgUCGalT1, is involved in anthocyanin galactosylation in purple celery. Celery is a well-known vegetable because of its rich nutrients, low calories, and medicinal values. Its petioles and leaf blades are the main organs acting as nutrient sources. UDP-galactose: cyanidin 3-O-galactosyltransferase can transfer the galactosyl moiety from UDP-galactose to the 3-O-position of cyanidin through glycosylation. This process enhances the stability and water solubility of anthocyanins...
March 8, 2018: Planta
Wen Zhu, Guihua Gong, Jie Pan, Shu Han, Wei Zhang, Youjia Hu, Liping Xie
Human serum albumin (HSA) has been extensively used in a series of clinical care settings for nearly seven decades. However, the broad application of this protein is seriously limited by its short supply. In this work, the codon sequence of HSA was cloned under the control of the alcohol oxidase 1 promoter (AOX1) and expressed as a secretory protein in Pichia pastoris. A recombinant strain displaying the highest HSA yield was selected by screening for resistance to the highest concentration of antibiotic G418...
March 5, 2018: Protein Expression and Purification
Sarah L Irons, Adam C Chambers, Olga Lissina, Linda A King, Robert D Possee
Baculovirus expression systems are well established as an easy and reliable way to produce high quality recombinant proteins. Baculoviruses can also be used to transduce mammalian cells, termed 'BacMam', with considerable potential in biomedical applications. This chapter explains the process of making a recombinant baculovirus, encompassing production of a recombinant virus by homologous recombination in insect cells, followed by amplification and titration of the virus-all steps needed before commencing gene expression and protein production...
February 21, 2018: Current Protocols in Protein Science
Merideth A Cooper, Joseph E Taris, Changhua Shi, David W Wood
In this work, we describe a novel self-cleaving tag technology based on a highly modified split-intein cleaving element. In this system, the N-terminal segment of an engineered split intein is expressed in E. coli and covalently immobilized onto a capture resin, while the smaller C-terminal intein segment is fused to the N-terminus of the desired target protein. The tagged target can then be expressed in an appropriate expression system, without concern for premature intein cleaving. During the purification, strong association between the intein segments effectively captures the tagged target onto the capture resin while simultaneously generating a cleaving-competent intein complex...
February 21, 2018: Current Protocols in Protein Science
Jeffrey A Lawton, Noelle A Prescott, Ping X Lawton
We have developed an integrated, project-oriented curriculum for undergraduate molecular biology and biochemistry laboratory courses spanning two semesters that is organized around the ldhA gene from the yogurt-fermenting bacterium Lactobacillus bulgaricus, which encodes the enzyme d-lactate dehydrogenase. The molecular biology module, which consists of nine experiments carried out over eleven sessions, begins with the isolation of genomic DNA from L. bulgaricus in yogurt and guides students through the process of cloning the ldhA gene into a prokaryotic expression vector, followed by mRNA isolation and characterization of recombinant gene expression levels using RT-PCR...
March 7, 2018: Biochemistry and Molecular Biology Education
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