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Affinity purification recombinant protein

Huihui Huang, Zheyan Wang, Zhenggen Yang, Zhen Li, Ping Wang, Wenqi Dong
Objective To construct eukaryotic expression vectors of human IgE heavy chain 2-4 region (IgECepsilon2-4) and purify the recombinant protein, and then capture its interacted proteins by surface plasmon resonance (SPR). Methods Three recombinant eukaryotic expression vectors of IgECepsilon2-4 containing different signal peptides were constructed and transiently transfected into HEK293FT suspension cells separately. The recombinant plasmid with the highest-level expression was selected to express the recombinant protein in a huge amount, and then the recombinant protein was purified by Ni-NTA affinity chromatography...
March 2018: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
Nagesh K Tripathi, Divyanshi Karothia, Ambuj Shrivastava, Swati Banger, Jyoti S Kumar
West Nile virus (WNV) is an emerging mosquito-borne virus which is responsible for severe and fatal encephalitis in humans and for which there is no licensed vaccine or therapeutic available to prevent infection. The envelope domain III protein (EDIII) of WNV was over-expressed in Escherichia coli and purified using a two-step chromatography process which included immobilized metal affinity chromatography and ion exchange chromatography. E. coli cells were grown in a bioreactor to high density using batch and fed-batch cultivation...
May 13, 2018: New Biotechnology
Samaneh Gholami, Nematolah Gheibi, Reza Falak, Koorosh Goodarzvand Chegini
Background: Betatrophin is a member of the angiopoietin-like (ANGPTL) family that has been implicated in both triglyceride and glucose metabolism. The physiological functions and molecular targets of this protein remain largely unknown; hence, a purified available protein would aid study of the exact role of betatrophin in lipid or glucose metabolism. Methods: In this study, we cloned the full-length cDNA of betatrophin from a human liver cDNA library. Betatrophin was expressed in the pET-21b- E...
April 2018: Reports of Biochemistry & Molecular Biology
Nidhi Puranik, N K Tripathi, V Pal, Ajay Kumar Goel
Surface array protein (Sap) can be an important biomarker for specific detection of Bacillus anthracis , which is released by the bacterium during its growth in culture broth. In the present work, we have cloned and expressed Sap in Escherichia coli . The culture conditions and cultivation media were optimized and used in batch fermentation process for scale up of Sap in soluble form. The recombinant Sap was purified employing affinity chromatography followed by diafiltration. The final yield of purified protein was 20 and 46 mg/l of culture during shake flasks and batch fermentation, respectively...
May 2018: 3 Biotech
Yanan Guo, Miao Yu, Na Jing, Shoutao Zhang
Embryonic stem cells and induced pluripotent stem cells depend on one of cytokines called leukemia inhibitory factor (LIF) to retain their undifferentiated state and pluripotency. Nevertheless, further progresses of stem cell scientific investigation and its possible application are limited owing to the expense of commercial LIF. Here we introduced a simple, practical and high level expression of MBP-mouse LIF through Escherichia coli system which was bioactive. The mLIF cDNA was inserted into vector of pMAL-C2X in order to generate N-terminal MBP-mLIF recombinant proteins in the cytoplasm of Escherichia coli...
May 11, 2018: Protein Expression and Purification
Philipp A M Schmidpeter, Xiaolong Gao, Vikrant Uphadyay, Jan Rheinberger, Crina M Nimigean
Cyclic nucleotide-modulated ion channels play several essential physiological roles. They are involved in signal transduction in photoreceptors and olfactory sensory neurons as well as pacemaking activity in the heart and brain. Investigations of the molecular mechanism of their actions, including structural and electrophysiological characterization, are restricted by the availability of stable, purified protein obtained from accessible systems. Here, we establish that SthK, a cyclic nucleotide-gated (CNG) channel from Spirochaeta thermophila , is an excellent model for investigating the gating of eukaryotic CNG channels at the molecular level...
May 11, 2018: Journal of General Physiology
Rui Li, Hongfang Ma, Longguang Jiang, Songlin Qiao, Yubao Zhi, Mingdong Huang, Ruiguang Deng, Gaiping Zhang
Scavenger receptors (SRs) play critical roles in various physiological and pathological pathways. One of them, CD163, is a multifunctional endocytic receptor and is characterized by a long-range scavenger receptor cysteine-rich (SRCR) repeat. However, the structural and functional details of this long-range SRCR repeat have not yet been elucidated. In this study, the CD163 long-range SRCR repeat was expressed in Drosophila Schneider 2 cells. The recombinant protein was homogeneous after purification by metal-affinity, cation-exchange and size-exclusion chromatography...
May 1, 2018: Acta Crystallographica. Section F, Structural Biology Communications
Ting Fang, Zhenghong Tao, Yanhong Liu, Changming Yu, Ruizhi Zhi, Rui Yu
CRM197 (cross-reacting material 197), a non-toxic mutant of diphtheria toxin, has wide application potential in biopharmaceuticals. However, it is difficult to express CRM197 in bacteria other than Corynebacterium diphtheriae. Here we proposed a new alternative method to produce soluble CRM197 without label in Escherichia coli. In particular, a synthetic gene coding for CRM197, optimized for E. coli codon usage, was cloned in the pET32a (+) vector. Accordingly, the over-expression of the protein was simply induced with IPTG in E...
April 25, 2018: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Dongdong Mu, Jiaojiao Lu, Mingqiang Qiao, Oscar P Kuipers, Jing Zhu, Xingjiang Li, Peizhou Yang, Yanyan Zhao, Shuizhong Luo, Xuefeng Wu, Shaotong Jiang, Zhi Zheng
Microbial transglutaminase (MTG) from Streptomyces mobaraensis has been widely used for crosslinking proteins in order to acquire products with improved properties. To improve the yield and enable a facile and efficient purification process, recombinant vectors, harboring various heterologous signal peptide-encoding fragments fused to the mtg gene, were constructed in Escherichia coli and then expressed in Bacillus subtilis. Signal peptides of both WapA and AmyQ (SP wapA and SP amyQ ) were able to direct the secretion of pre-pro-MTG into the medium...
April 25, 2018: Applied Microbiology and Biotechnology
Kristell Lebozec, Martine Jandrot-Perrus, Gilles Avenard, Olivier Favre-Bulle, Philippe Billiald
Monoclonal antibody fragments (Fab) are a promising class of therapeutic agents. Fabs are aglycosylated proteins and so many expression platforms have been developed including prokaryotic, yeast and mammalian cells. However, these platforms are not equivalent in terms of cell line development and culture time, product quality and possibly cost of production that greatly influence the success of a drug candidate's pharmaceutical development. This study is an assessment of the humanized Fab fragment ACT017 produced from two microorganisms (Escherichia coli and Pichia pastoris) and one mammalian cell host (CHO)...
April 21, 2018: New Biotechnology
Md Nasimuzzaman, Johannes C M van der Loo, Punam Malik
Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a promising vector for gene therapy application. Here, baculovirus is produced by transient transfection of the baculovirus plasmid DNA (bacmid) in an adherent culture of Sf9 cells. Baculovirus is subsequently expanded in Sf9 cells in a serum-free suspension culture until the desired volume is obtained. It is then purified from the culture supernatant using heparin affinity chromatography...
April 9, 2018: Journal of Visualized Experiments: JoVE
Juan-Juan Chen, Si-Yong Huang, Peng-Fei Ma, Bi-Jia Wu, Si-Lang Zhou, Yong-Xing Zhao, Jun-Mei Gong, Ying-Min Liang
OBJECTIVE: To express and purify the mouse endothelial cell-targeted recombinant Notch ligand protein mD1R, and to investigate its effect on hematopoiesis after carbon tetrachloride damage. METHODS: PCR was performed to clone and construct the expression vector pET22b(+)-mD1R. The mD1R successfully transformed into E. coli was induced by IPTG, and purified with Ni2+ -beads affinity chromatography. The target protein was detected by SDS-PAGE. The fluorescence-activated cell sorting analysis (FACS), cell adhesion test, immunofluorescence staining and quantitative real-time PCR were employed to detect the endothelial cell-targeted and Notch signaling-activated biological characteristics of mD1R...
April 2018: Zhongguo Shi Yan Xue Ye Xue za Zhi
Shabih Shakeel, James D Evans, Mark Hazelbaker, C Cheng Kao, Robert C Vaughan, Sarah J Butcher
Human parechoviruses (HPeV) are picornaviruses with a highly-ordered RNA genome contained within icosahedrally-symmetric capsids. Ordered RNA structures have recently been shown to interact with capsid proteins VP1 and VP3 and facilitate virus assembly in HPeV1. Using an assay that combines reversible cross-linking, RNA affinity purification and peptide mass fingerprinting (RCAP), we mapped the RNA-interacting regions of the capsid proteins from the whole HPeV1 virion in solution. The intrinsically-disordered N-termini of capsid proteins VP1 and VP3, and unexpectedly, VP0, were identified to interact with RNA...
April 11, 2018: Scientific Reports
Vladislav V Mokhonov, Ekaterina A Vasilenko, Ekaterina N Gorshkova, Irina V Astrakhantseva, Dmitry V Novikov, Viktor V Novikov
Binding of native bacterial protein SlyD to metal affinity matrices remains a major problem in affinity purification of His-tagged recombinant proteins from Escherichia coli cells. In this study, four novel E. coli strains that lack the expression of SlyD/SlyX, were engineered using λ-red mediated chromosomal deletion. The resultant mutant E. coli strains allow us to obtain SlyD-free proteins immediately after metal affinity chromatography, and eliminate additional purification processes. As a model protein, bispecific antibodies composed of anti-F4/80 VH H module and anti-TNF VH H module (MYSTI-2) were used...
April 4, 2018: Biochemical and Biophysical Research Communications
William S Kish, Matthew K Roach, Hiroyuki Sachi, Amith D Naik, Stefano Menegatti, Ruben G Carbonell
Prior work described the identification and characterization of erythropoietin-binding cyclic peptides SLFFLH, VVFFVH, FSLLHH and FSLLSH (all of the form cyclo[(Nα -Ac)Dap(A)-X1 -X6 -AE], wherein X1 -X6 is the listed sequences). In this work, the peptide ligands were synthesized on Toyopearl chromatographic resins and utilized for purifying recombinant human erythropoietin (rHuEPO) from complex sources. Elution buffer pH and composition were optimized to maximize the recovery of standard rHuEPO from the peptide resins...
March 27, 2018: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
J Wang, M Liu, X Bai, H Zhang, Z Xu, D Zhao, Y Zhao
Thymosin beta 10 (Tβ10) is a member of the β-thymosin family. As an actin-binding peptide, thymosin β10 is involved in many important biological activities. Transcriptome sequencing results suggest that Tβ10 may play important roles in the growth of deer antler. In this study, Tβ10 cDNA was isolated from sika deer, and complete open reading frame consisting of 129 nucleotides was obtained by PCR amplification. The predicted peptide was 42 amino acids in length. The sdTβ10 cDNA was cloned and expressed in Escherichia coli resulting in a 6 kDa recombinant-His tagged protein...
December 2017: Polish Journal of Veterinary Sciences
San Jun Guo, Shan Ren, Yong En Xie
Acinetobacter baumannii (A. Baumannii) is an emerging opportunistic pathogen responsible for hospital-acquired infections, and which now constitutes a sufficiently serious threat to public health to necessitate the development of an effective vaccine. In this study, a recombinant fused protein named OmpK/Omp22 and two individual proteins OmpK and Omp22 were obtained using recombinant expression and Ni-affinity purification. Groups of BALB/c mice were immunized with these proteins and challenged with a clinically isolated strain of A...
February 2018: Biomedical and Environmental Sciences: BES
Matthew J Schellenberg, Robert M Petrovich, Christine C Malone, R Scott Williams
Recombinant protein expression systems that produce high yields of pure proteins and multi-protein complexes are essential to meet the needs of biologists, biochemists, and structural biologists using X-ray crystallography and cryo-electron microscopy. An ideal expression system for recombinant human proteins is cultured human cells where the correct translation and chaperone machinery are present. However, compared to bacterial expression systems, human cell cultures present several technical challenges to their use as an expression system...
March 25, 2018: Protein Science: a Publication of the Protein Society
Marisa I Mendes, Mariana Gutierrez Salazar, Kether Guerrero, Isabelle Thiffault, Gajja S Salomons, Laurence Gauquelin, Luan T Tran, Diane Forget, Marie-Soleil Gauthier, Quinten Waisfisz, Desiree E C Smith, Cas Simons, Marjo S van der Knaap, Iris Marquardt, Aida Lemes, Hanna Mierzewska, Bernhard Weschke, Wolfgang Koehler, Benoit Coulombe, Nicole I Wolf, Geneviève Bernard
Hypomyelinating leukodystrophies are genetic disorders characterized by insufficient myelin deposition during development. They are diagnosed on the basis of both clinical and MRI features followed by genetic confirmation. Here, we report on four unrelated affected individuals with hypomyelination and bi-allelic pathogenic variants in EPRS, the gene encoding cytoplasmic glutamyl-prolyl-aminoacyl-tRNA synthetase. EPRS is a bifunctional aminoacyl-tRNA synthetase that catalyzes the aminoacylation of glutamic acid and proline tRNA species...
April 5, 2018: American Journal of Human Genetics
Tanja Bartoschik, Stefanie Galinec, Christian Kleusch, Katarzyna Walkiewicz, Dennis Breitsprecher, Sebastian Weigert, Yves A Muller, Changjiang You, Jacob Piehler, Thomas Vercruysse, Dirk Daelemans, Nuska Tschammer
MicroScale Thermophoresis (MST) is a frequently used method for the quantitative characterization of intermolecular interactions with several advantages over other technologies. One of these is its capability to determine equilibrium constants in solution including complex biological matrices such as cell lysates. MST requires one binding partner to be fluorescent, which is typically achieved by labeling target proteins with a suitable fluorophore. Here, we present a near-native, site-specific in situ labeling strategy for MST experiments that enables reliable measurements in cell lysates and that has distinct advantages over routine covalent labeling techniques...
March 21, 2018: Scientific Reports
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