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Protein expression and purification

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https://www.readbyqxmd.com/read/28535613/expression-and-purification-of-biologically-active-human-bone-morphogenetic-protein-4-in-recombinant-chinese-hamster-ovary-cells
#1
Minyub Cha, Nara Han, Jia Pi, Yongsu Jeong, Kwanghee Baek, Jaeseung Yoon
Bone morphogenetic protein-4 (BMP-4) is considered to have therapeutic potential for various diseases including cancers; however, the high expression of biologically active recombinant human BMP-4 (rhBMP-4) needed to manufacture rhBMP-4 for therapeutic purposes has yet to be established. In the current study, we established a recombinant Chinese hamster ovary (rCHO) cell line overexpressing rhBMP-4 as well as a production process using 7.5-l bioreactor (5 l working volume). The expression of the mature rhBMP-4 was significantly enhanced by recombinant furin expression...
May 24, 2017: Journal of Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28534479/efficient-protein-production-inspired-by-how-spiders-make-silk
#2
Nina Kronqvist, Médoune Sarr, Anton Lindqvist, Kerstin Nordling, Martins Otikovs, Luca Venturi, Barbara Pioselli, Pasi Purhonen, Michael Landreh, Henrik Biverstål, Zigmantas Toleikis, Lisa Sjöberg, Carol V Robinson, Nicola Pelizzi, Hans Jörnvall, Hans Hebert, Kristaps Jaudzems, Tore Curstedt, Anna Rising, Jan Johansson
Membrane proteins are targets of most available pharmaceuticals, but they are difficult to produce recombinantly, like many other aggregation-prone proteins. Spiders can produce silk proteins at huge concentrations by sequestering their aggregation-prone regions in micellar structures, where the very soluble N-terminal domain (NT) forms the shell. We hypothesize that fusion to NT could similarly solubilize non-spidroin proteins, and design a charge-reversed mutant (NT*) that is pH insensitive, stabilized and hypersoluble compared to wild-type NT...
May 23, 2017: Nature Communications
https://www.readbyqxmd.com/read/28529565/prodelphinidins-isolated-from-chinese-bayberry-leaves-induces-apoptosis-via-the-p53-dependent-signaling-pathways-in-ovcar-3-human-ovarian-cancer-cells
#3
Yu Fu, Xingqian Ye, Malcolm Lee, Gary Rankin, Yi Charlie Chen
Chinese bayberry leaves are rich in prodelphinidins. Since the isolation and purification of prodelphinidins is difficult, the association between the degree of prodelphinidin polymerization and their anti-carcinogenic activity remains ambiguous. The cytotoxic and apoptotic activities of prodelphinidin Chinese bayberry leaf extracts (PCBLs), oligomeric proanthocyanidins (OPAs) and polymeric proanthocyanidins (PPAs), isolated by normal-phase preparative high-performance liquid chromatography were investigated in OVCAR-3 human ovarian cancer cells...
May 2017: Oncology Letters
https://www.readbyqxmd.com/read/28526409/expression-and-purification-of-functional-pdgf-receptor-beta
#4
Qingbin Shang, Liang Zhao, Xiaojing Wang, Meimei Wang, Sen-Fang Sui, Li-Zhi Mi
Platelet Derived Growth Factor receptors (PDGFRs), members of receptor tyrosine kinase superfamily, play essential roles in early hematopoiesis, angiogenesis and organ development. Dysregulation of PDGF receptor signaling under pathological conditions associates with cancers, vascular diseases, and fibrotic diseases. Therefore, they are attractive targets in drug development. Like any other membrane proteins with a single-pass transmembrane domain, the high-resolution structural information of the full-length PDGF receptors is still not resolved...
May 16, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/28522394/interaction-research-on-an-antiviral-molecule-that-targets-the-coat-protein-of-southern-rice-black-streaked-dwarf-virus
#5
Longlu Ran, Yan Ding, Liangzhi Luo, Xiuhai Gan, Xiangyang Li, Yongzhong Chen, Deyu Hu, Baoan Song
Southern rice black-streaked dwarf virus (SRBSDV) coat protein (P10) is the key protein required for viral transmission and host plant infection and is thus a promising target for anti-SRBSDV agent screening. In this study, P10 was obtained from Escherichia coli through cloning, expression, and purification. The antiviral agent Ningnanmycin was selected as control, and a series of antiviral compounds based on the structural scaffold of ferulic acid were analyzed. Size-exclusion chromatography analysis results showed that compound F27 can alter the aggregation of P10 proteins...
May 15, 2017: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/28512649/purification-and-characterization-of-schwann-cells-from-adult-human-skin-and-nerve
#6
Jo Anne Stratton, Ranjan Kumar, Sarthak Sinha, Prajay Shah, Morgan Stykel, Yuval Shapira, Rajiv Midha, Jeff Biernaskie
Despite its modest capacity for regeneration, peripheral nervous system injury often results in significant long-term disability. Supplementing peripheral nervous system injury with autologous Schwann cells (SCs) may serve to rejuvenate the postinjury environment to enhance regeneration and ultimately improve functional outcomes. However, human nerve-derived SC (hN-SC) collection procedures require invasive surgical resection. Here, we describe the characterization of SCs from adult human skin (hSk-SCs) of four male donors ranging between 27 and 46 years old...
May 2017: ENeuro
https://www.readbyqxmd.com/read/28510356/purification-of-recombinant-a%C3%AE-1-42-and-pglu-a%C3%AE-3-42-using-preparative-sds-page
#7
Claudia Spahn, Michael Wermann, Rico Eichentopf, Gerd Hause, Dagmar Schlenzig, Stephan Schilling
Recombinant expression and purification of amyloid peptides represents a common basis for investigating the molecular mechanisms of amyloid formation and toxicity. However, the isolation of the recombinant peptides is hampered by inefficient separation from contaminants such as the fusion protein required for efficient expression in E. coli. Here, we present a new approach for the isolation of highly purified Aβ(1-42) and pGlu-Aβ(3-42), which is based on a separation using preparative SDS-PAGE. The method relies on the purification of the Aβ fusion protein by affinity chromatography followed by preparative SDS-PAGE under reducing conditions and subsequent removal of detergents by precipitation...
May 16, 2017: Electrophoresis
https://www.readbyqxmd.com/read/28509348/reconstitution-of-the-plant-ubiquitination-cascade-in-bacteria-using-a-synthetic-biology-approach
#8
Yufang Han, Jianhang Sun, Jun Yang, Zhaoyun Tan, Jijing Luo, Dongping Lu
Ubiquitination modulates nearly all aspects of plant life. Here, we reconstituted the Arabidopsis thaliana ubiquitination cascade in Escherichia coli using a synthetic biology approach. In this system, plant proteins are expressed and then immediately participate in ubiquitination reactions within E. coli cells. Additionally, the purification of the individual ubiquitination components prior to setting up the ubiquitination reactions is skipped. To establish the reconstituted system, we co-expressed Arabidopsis ubiquitin (Ub) and ubiquitination substrates with E1, E2 and E3 enzymes in E...
May 16, 2017: Plant Journal: for Cell and Molecular Biology
https://www.readbyqxmd.com/read/28508361/photoaffinity-labeling-of-pentameric-ligand-gated-ion-channels-a-proteomic-approach-to-identify-allosteric-modulator-binding-sites
#9
Selwyn S Jayakar, Gordon Ang, David C Chiara, Ayman K Hamouda
Photoaffinity labeling techniques have been used for decades to identify drug binding sites and to study the structural biology of allosteric transitions in transmembrane proteins including pentameric ligand-gated ion channels (pLGIC). In a typical photoaffinity labeling experiment, to identify drug binding sites, UV light is used to introduce a covalent bond between a photoreactive ligand (which upon irradiation at the appropriate wavelength converts to a reactive intermediate) and amino acid residues that lie within its binding site...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28506354/-immunoprotective-effect-of-combined-pneumococcal-endopeptidase-o-and-pneumococcal-surface-adhesin-a-vaccines-against-streptococcus-pneumoniae-infection
#10
Jing Zhang, Ya-Li Cui, Yong-Mei Jiang
OBJECTIVE: To investigate the prokaryotic expression of proteins pneumococcal endopeptidase O (PepO) and pneumococcal surface adhesin A (PsaA) in Streptococcus pneumoniae and their immunoprotective effect as vaccine candidate proteins. METHODS: Specific primers of target gene fragments were designed, and then PCR amplification was performed to establish recombinant plasmids pET28a(+)-pepO and pET28a(+)-psaA, which were transformed into host cells, Escherichia coli BL21 and DE3, respectively, to induce expression...
May 2017: Zhongguo Dang Dai Er Ke za Zhi, Chinese Journal of Contemporary Pediatrics
https://www.readbyqxmd.com/read/28501598/microgramma-vacciniifolia-polypodiaceae-fronds-contain-a-multifunctional-lectin-with-immunomodulatory-properties-on-human-cells
#11
Leydianne Leite de Siqueira Patriota, Thamara Figueiredo Procópio, Jéssica de Santana Brito, Virginie Sebag, Ana Patrícia Silva de Oliveira, Ana Karine de Araújo Soares, Leyllane Rafael Moreira, Thâmarah de Albuquerque Lima, Tatiana Soares, Túlio Diego da Silva, Patrícia Maria Guedes Paiva, Virgínia Maria Barros de Lorena, Cristiane Moutinho Lagos de Melo, Lidiane Pereira de Albuquerque, Thiago Henrique Napoleão
In this study, we report the purification and characterization of a multifunctional lectin (MvFL) from Microgramma vacciniifolia fronds as well as its immunomodulatory properties on human peripheral blood mononuclear cells (PBMCs). MvFL (pI 4.51; 54kDa) is a glycoprotein able to inhibit trypsin activity and that has sequence similarities (32% coverage) with a plant RNA-binding protein. Hemagglutinating activity of MvFL was not altered by heating at 100°C for 30min, but was reduced in alkaline pH (8.0 and 9...
May 10, 2017: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/28490711/purification-crystallization-and-x-ray-diffraction-study-of-the-c-terminal-domain-of-human-herpesvirus-6a-immediate-early-protein-2
#12
Junjie Wang, Mitsuhiro Nishimura, Aika Wakata, Yasuko Mori
Human herpesvirus 6A (HHV-6A) starts its replication cycle following the action of immediate early proteins that transactivate viral promoters. Immediate early protein 2 (IE2) of HHV-6A is a 1500 amino acid polypeptide with a C-terminal region that is conserved among beta-herpesvirus subfamily members. In this study, a structural domain in the homologous C-terminal region was subjected to secondary structure prediction, and residues 1324-1500 were subsequently designated as the C-terminal domain of IE2 (IE2-CTD)...
April 7, 2017: Kobe Journal of Medical Sciences
https://www.readbyqxmd.com/read/28490692/-identification-of-the-interacting-proteins-with-s100a8-or-s100a9-by-affinity-purification-and-mass-spectrometry
#13
Jing Wang, Xuemei Zhang, Zheng Li, Xiayu Li, Jian Ma, Shourong Shen
To identify the interacting proteins with S100A8 or S100A9 in HEK293 cell line by flag-tag affinity purification and liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS).
 Methods: The p3×Flag-CMV-S100A8 and p3×Flag-CMV-S100A9 expression vectors were constructed by inserting S100A8 or S100A9 coding sequence. The recombinant plasmids were then transfected into HEK293 cells. Affinity purification and LC-MS/MS were applied to identify the proteins interacting with S100A8 or S100A9. Bioinformatics analysis was used to seek the gene ontology of the interacting proteins...
April 28, 2017: Zhong Nan da Xue Xue Bao. Yi Xue Ban, Journal of Central South University. Medical Sciences
https://www.readbyqxmd.com/read/28490597/molluscum-contagiosum-virus-protein-mc005-inhibits-nf%C3%AE%C2%BAb-activation-by-targeting-nemo-regulated-ikk-activation
#14
Gareth Brady, Darya A Haas, Paul J Farrell, Andreas Pichlmair, Andrew G Bowie
Molluscum Contagiosum Virus (MCV), the only known extant, human-adapted poxvirus, causes a long-duration infection characterized by skin lesions that typically display an absence of inflammation despite containing high titres of live virus. Despite this curious presentation, MCV is very poorly characterized in terms of host-pathogen interactions. The absence of inflammation around MCV lesions suggests the presence of potent inhibitors of human anti-viral immunity and inflammation. However, only a small number of MCV immunomodulatory genes have been characterized in detail...
May 10, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28487982/post-translational-modifications-and-their-applications-in-eye-research-review
#15
Bing-Jie Chen, Thomas Chuen Lam, Long-Qian Liu, Chi-Ho To
Gene expression is the process by which genetic information is used for the synthesis of a functional gene product, and ultimately regulates cell function. The increase of biological complexity from genome to proteome is vast, and the post-translational modification (PTM) of proteins contribute to this complexity. The study of protein expression and PTMs has attracted attention in the post‑genomic era. Due to the limited capability of conventional biochemical techniques in the past, large‑scale PTM studies were technically challenging...
June 2017: Molecular Medicine Reports
https://www.readbyqxmd.com/read/28487964/effects-of-the-cyclophilin-type-peptidylprolyl-cis-trans-isomerase-from-pyropia-yezoensis-against-hydrogen-peroxide-induced-oxidative-stress-in-hepg2-cells
#16
Eun-Young Kim, Youn Hee Choi, Chang Geun Choi, Taek-Jeong Nam
The present study aimed to describe the expression and purification of cyclophilin-type peptidylprolyl cis-trans isomerase (PPI) from the red alga Pyropia yezoensis. The antioxidant activity of the purified protein was also demonstrated, based on its ability to act against oxidative stress in HepG2 human hepatocellular carcinoma cells. HepG2 cells that were treated with recombinant PPI protein exhibited a reduction in the formation of hydrogen peroxide (H2O2)‑mediated reactive oxygen species (ROS). In HepG2 cells, treatment of recombinant PPI protein expression diminished H2O2‑mediated oxidative stress and restored both the expression and the activity of certain antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and thioredoxin reductase (TRR)...
June 2017: Molecular Medicine Reports
https://www.readbyqxmd.com/read/28487257/functional-efficacy-of-human-recombinant-fgf-2s-tagged-with-his-6-and-his-asn-6-at-the-n-and-c-termini-in-human-gingival-fibroblast-and-periodontal-ligament-derived-cells
#17
Ji-Hye Lee, Ji-Eun Lee, Kyung-Jung Kang, Young-Joo Jang
Fibroblast growth factor (FGF) is a multifunctional growth factor that induces cell proliferation, survival, migration, and differentiation in various cell types and tissues. With these biological functions, FGF-2 has been evaluated for clinical use in the regeneration of damaged tissues. The expression of hFGF-2 in Escherichia coli and a purification system using the immobilized metal affinity chromatography (IMAC) is well established to generate a continuous supply of FGF-2. Although hexa-histidine tag (H6) is commonly used for IMAC purification, hexa-histidine-asparagine tag (HN6) is also efficient for purification as it is easily exposed on the surface of the protein...
May 6, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28485212/purification-and-characterization-of-hemolysin-from-periodontopathogenic-bacterium-eikenella-corrodens-strain-1073
#18
Fariha Jasin Mansur, Sari Takahara, Mihoko Yamamoto, Masafumi Shimatani, Mohammad Minnatul Karim, Yuichiro Noiri, Shigeyuki Ebisu, Hiroyuki Azakami
Eikenella corrodens 1073 was found to show hemolytic activity when grown on sheep blood agar. A high and dose-dependent hemolytic activity was detected in the cell envelope fraction, which was further purified by ion-exchange and gel-filtration chromatography. Consequently, a 65-kDa protein with hemolytic activity was obtained, suggesting that this protein might be a hemolysin. Its N-terminal amino acid sequence was nearly identical to that of X-prolyl aminopeptidase from E. corrodens ATCC 23834. To confirm that X-prolyl aminopeptidase functions as a hemolytic factor, we expressed the hlyA gene, encoding X-prolyl aminopeptidase, in Escherichia coli...
June 2017: Bioscience, Biotechnology, and Biochemistry
https://www.readbyqxmd.com/read/28484256/improved-thermostability-of-an-acidic-xylanase-from-aspergillus-sulphureus-by-combined-disulphide-bridge-introduction-and-proline-residue-substitution
#19
Wenhan Yang, Yongzhi Yang, Lingdi Zhang, Hang Xu, Xiaojing Guo, Xu Yang, Bing Dong, Yunhe Cao
As a feed additive, xylanase has been widely applied in the feed of monogastric animals, which contains multiple plant polysaccharides. However, during feed manufacture, the high pelleting temperatures challenge wild-type xylanases. The aim of this study was to improve the thermostability of Aspergillus sulphureus acidic xylanase. According to the predicted protein structure, a series of disulphide bridges and proline substitutions were created in the xylanase by PCR, and the mutants were expressed in Pichia pastoris...
May 8, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28480316/expression-and-purification-of-mini-g-proteins-from-escherichia-coli
#20
Byron Carpenter, Christopher G Tate
Heterotrimeric G proteins modulate intracellular signalling by transducing information from cell surface G protein-coupled receptors (GPCRs) to cytoplasmic effector proteins. Structural and functional characterisation of GPCR-G protein complexes is important to fully decipher the mechanism of signal transduction. However, native G proteins are unstable and conformationally dynamic when coupled to a receptor. We therefore developed an engineered minimal G protein, mini-Gs, which formed a stable complex with GPCRs, and facilitated the crystallisation and structure determination of the human adenosine A2A receptor (A2AR) in its active conformation...
April 20, 2017: Bio-protocol
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