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Protein Expression and Purification

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https://www.readbyqxmd.com/read/28438686/expression-purification-and-molecular-characterization-of-a-novel-endoglucanase-protein-from-bacillus-subtilis-sb13
#1
Xuefang Guan, Penglian Chen, Qingxian Xu, Lei Qian, Juqing Huang, Bin Lin
Bacillus subtilis strain SB13 which is isolated in our previous work was confirmed to produce endoglucanase. In this study, a novel endoglucanase gene (accession number: KX576676) was identified and cloned from SB13. Compared with other consensus sequence of reported endoglucanase genes in the GenBank database, this gene displays five differences (including T740C,A874G,A983G, T1210G and T1301C), which leading to five amino acid changes. Homology modeling has indicated that these five changes were located in the α-helix and random coil regions of the glycosyl hydrolase family 5 (GH5) domain, the random coil and β-sandwich of the type 3 carbohydrate-binding module (CBM3) domain, and the random coil domain...
April 21, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28428153/development-of-polyol-responsive-antibody-mimetics-for-single-step-protein-purification
#2
Richard J Suderman, Daren A Rice, Shane D Gibson, Eric J Strick, David M Chao
The purification of functional proteins is a critical pre-requisite for many experimental assays. Immunoaffinity chromatography, one of the fastest and most efficient purification procedures available, is often limited by elution conditions that disrupt structure and destroy enzymatic activity. To address this limitation, we developed polyol-responsive antibody mimetics, termed nanoCLAMPs, based on a 16 kDa carbohydrate binding module domain from Clostridium perfringens hyaluronidase. nanoCLAMPs bind targets with nanomolar affinity and high selectivity yet release their targets when exposed to a neutral polyol-containing buffer, a composition others have shown to preserve quaternary structure and enzymatic activity...
April 17, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28419821/expression-of-the-enzymatically-active-legumain-like-cysteine-proteinase-tvlegu-1-of-trichomonas-vaginalis-in-pichia-pastoris
#3
Gerardo Reséndiz-Cardiel, Rossana Arroyo, Jaime Ortega-López
The legumain-like cysteine proteinase TvLEGU-1 from Trichomonas vaginalis plays a major role in trichomonal cytoadherence. However, its structure-function characterization has been limited by the lack of a reliable recombinant expression platform to produce this protein in its native folded conformation. TvLEGU-1 has been expressed in Escherichia coli as inclusion bodies and all efforts to refold it have failed. Here, we describe the expression of the synthetic codon-optimized tvlegu-1 (tvlegu-1-opt) gene in Pichia pastoris strain X-33 (Mut+) under the inducible AOX1 promoter...
April 15, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28414067/effective-strategies-for-host-cell-protein-clearance-in-downstream-processing-of-monoclonal-antibodies-and-fc-fusion-proteins
#4
REVIEW
Yifeng Li
Recombinant therapeutic proteins are typically produced through cell culture process. Host cell proteins (HCPs) are endogenous proteins derived from the host cells used for such bioproduction. HCPs form a major class of process-related impurities and even at low levels they can potentially compromise the safety and efficacy of biopharmaceuticals. Therefore, they need to be adequately removed via the downstream process. HCPs are complex mixtures with diverse physiochemical properties, and certain subpopulations can bind to the intended product...
April 13, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28410993/development-of-a-novel-recombinant-lhrh-fusion-protein-for-therapy-of-androgen-and-estrogen-dependent-cancers
#5
Jagdish C Gupta, Rohit S Hada, P Sahai, G P Talwar
LHRH based vaccines are promising candidates for therapy of androgen and estrogen dependent cancers. We report in this communication development of a novel recombinant protein vaccine candidate against LHRH. A synthetic gene was designed in which the codon sequence in the LHRH decapeptide was modified by substituting the codon for 6-glycine with that of l-leucine. Further the LHRH(6leu) gene was linked to heat-labile enterotoxin of E. coli (LTB) as carrier. This LHRH(6leu)-LTB gene was cloned into a prokaryotic expression vector under the control of inducible and strong bacteriophage T7 promoter to over-express LHRH(leu) fused to LTB as recombinant protein in E...
April 11, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28400295/phic31-integrase-can-improve-the-efficiency-of-different-construct-designs-for-monoclonal-antibody-expression-in-cho-cells
#6
Maryam Ahmadi, Fereidoun Mahboudi, Samira Ahmadi, Saeedeh Ebadat, Fatemeh Nematpour, Mohammad Reza Akbari Eidgahi, Fatemeh Davami
OBJECTIVES: Several types of expression vectors have been used for recombinant protein expression in Chinese hamster ovary cells (CHO) which usually result in variable and unstable levels of expression. METHODS AND RESULTS: In this study, we have compared the mAb0014 expression level of single ORF/IRES vector and dual ORF vector in the presence and absence of phiC31 integrase targeting system. Both expression vectors contain an elongation factor 1α (EF1α) promoter upstream of LC and harboring an attB site...
April 9, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28400296/recombinant-expression-and-purification-of-the-rna-binding-larp6-proteins-from-fish-genetic-model-organisms
#7
José M Castro, Daniel A Horn, Karen A Lewis
The RNA-binding proteins that comprise the La-related protein (LARP) superfamily have been implicated in a wide range of cellular functions, from tRNA maturation to regulation of protein synthesis. To more expansively characterize the biological function of the LARP6 subfamily, we have recombinantly expressed the full-length LARP6 proteins from two teleost fish, platyfish (Xiphophorus maculatus) and zebrafish (Danio rerio). The yields of the recombinant proteins were enhanced to >2 mg/L using a tandem approach of an N-terminal His6-SUMO tag and an iterative solubility screening assay to identify structurally stabilizing buffer components...
April 8, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28392342/exploitation-of-human-cd99-expressing-mouse-myeloma-cells-as-immunogen-for-production-of-mouse-specific-polyclonal-antibodies
#8
Nuchjira Takheaw, Witida Laopajon, Kantinan Chuensirikulchai, Watchara Kasinrerk, Supansa Pata
In this study, we describe the application of a molecular biology technique for the production of mouse polyclonal antibodies (pAbs) specific to human cell surface molecules. Production of the pAb specific to the human CD99 surface molecule was used as the study model. The retroviral expression system was employed to generate human CD99 expressing mouse myeloma cells. After cell sorting and single cell cloning, a myeloma clone which stably expressed high levels of human CD99 on its surface was established. The human CD99 expressing mouse myeloma cells were then used as the immunogen for immunization of BALB/c mice...
April 6, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28389350/process-development-for-production-and-purification-of-the-schistosoma-mansoni-sm14-antigen
#9
Leonardo Damasceno, Gerd Ritter, Carl A Batt
The trematode Schistosoma mansoni Sm14 antigen was expressed in the yeast Pichia pastoris and secreted into the culture medium at yields of approximately 250 mg L(-1). Sm14 belongs to a family of fatty-acid binding proteins and appears to play an important role in uptake, transport, and compartmentalization of lipids in S. mansoni and it is a potential vaccine candidate in both humans and domesticated animals. The Sm14 gene was codon-optimized for expression in P. pastoris, and placed under transcription of the strong methanol inducible AOX1 promoter...
April 5, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28385582/an-easy-method-for-bacterial-expression-and-purification-of-wild-type-and-mutant-superoxide-dismutase-1-sod1
#10
Babila J Tachu, Katharina A Wüsten, Maria C Garza, Holger Wille, Gültekin Tamgüney
No abstract text is available yet for this article.
April 3, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28347752/expression-purification-and-characterization-of-active-untagged-recombinant-human-leukemia-inhibitory-factor-from-e-coli
#11
Xueyan Xi, Xiaolu Li, Fan Wu, Xin Guan, Lan Jin, Yang Guo, Wei Song, Boyu Du
Leukemia inhibitory factor (LIF), a member of IL-6 cytokine family, is considered to be a pleiotropic cytokine and function both in cellular proliferation and differentiation. It had been widely used in biomedical research. The large requirement for this cytokine led to the continuing development of its efficient production methods. Due to its low expression and purification yields when it was produced in eukaryotic cells, recombinant human LIF had always been expressed either as inclusion body or as fusion protein in E coli...
March 24, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28343996/optimization-of-overexpression-of-a-chaperone-protein-of-steroid-c25-dehydrogenase-for-biochemical-and-biophysical-characterization
#12
Ewa Niedzialkowska, Beata Mrugała, Agnieszka Rugor, Mateusz P Czub, Anna Skotnicka, Julien J H Cotelesage, Graham N George, Maciej Szaleniec, Wladek Minor, Krzysztof Lewiński
Molybdenum is an essential nutrient for metabolism in plant, bacteria, and animals. Molybdoenzymes are involved in nitrogen assimilation and oxidoreductive detoxification, and bioconversion reactions of environmental, industrial, and pharmaceutical interest. Molybdoenzymes contain a molybdenum cofactor (Moco), which is a pyranopterin heterocyclic compound that binds a molybdenum atom via a dithiolene group. Because Moco is a large and complex compound deeply buried within the protein, molybdoenzymes are accompanied by private chaperone proteins responsible for the cofactor's insertion into the enzyme and the enzyme's maturation...
March 23, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28342833/a-high-density-cho-s-transient-transfection-system-comparison-of-expicho-and-expi293
#13
Nina K Jain, Susan Barkowski-Clark, Richard Altman, Krista Johnson, Fang Sun, Jonathan Zmuda, Chao Yan Liu, Adriana Kita, Ryan Schulz, Alyssa Neill, Robert Ballinger, Rekha Patel, Jian Liu, Alinafe Mpanda, Brian Huta, Henry Chiou, Walter Voegtli, Tadas Panavas
Chinese Hamster Ovary (CHO) cells are the principal mammalian host used for stable cell line generation and biotherapeutic protein production. Until recently, production of milligrams to grams of protein in CHO transient systems was challenging. As such, Human Embryonic Kidney (HEK293) cells are the most common mammalian cell type used for transient transfection. The post-translational modifications (PTMs) of a protein are dictated in part by the cell line used for expression, and changes in PTMs have been shown to affect both the activity and biophysical properties of proteins...
March 23, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28336201/a-facile-method-for-isolation-of-recombinant-human-apolipoprotein-a-i-from-e-%C3%A2-coli
#14
Nikita Ikon, Jennifer Shearer, Jianfang Liu, Jesse J Tran, ShiBo Feng, Ayako Kamei, Jennifer A Beckstead, Robert S Kiss, Paul M Weers, Gang Ren, Robert O Ryan
Apolipoprotein (apo) A-I is the major protein component of high-density lipoprotein (HDL) and plays key roles in the Reverse Cholesterol Transport pathway. In the past decade, reconstituted HDL (rHDL) has been employed as a therapeutic agent for treatment of atherosclerosis. The ability of rHDL to promote cholesterol efflux from peripheral cells has been documented to reduce the size of atherosclerotic plaque lesions. However, development of apoA-I rHDL-based therapeutics for human use requires a cost effective process to generate an apoA-I product that meets "Good Manufacturing Practice" standards...
March 20, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28330825/streamlining-workflow-and-automation-to-accelerate-laboratory-scale-protein-production
#15
REVIEW
Jennifer Konczal, Christopher H Gray
Protein production facilities are often required to produce diverse arrays of proteins for demanding methodologies including crystallography, NMR, ITC and other reagent intensive techniques. It is common for these teams to find themselves a bottleneck in the pipeline of ambitious projects. This pressure to deliver has resulted in the evolution of many novel methods to increase capacity and throughput at all stages in the pipeline for generation of recombinant proteins. This review aims to describe current and emerging options to accelerate the success of protein production in Escherichia coli...
March 19, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28323169/isolation-and-characterization-of-human-capg-expressed-and-post-translationally-modified-in-pichia-pastoris
#16
Agnes Papala, Marc Sylvester, Nadine Dyballa-Rukes, Sabine Metzger, Jochen D'Haese
CapG is an actin-binding protein, which is overexpressed in a variety of tumors, i.e. breast, ovarian, pancreatic and lung carcinoma. We successfully expressed human CapG in the wild type strain X-33 of the methylotrophic yeast Pichia pastoris (P. pastoris), which does not express endogenous CapG, in order to characterize this protein in more detail. After mechanical cell lysis, debris was centrifuged and the soluble protein was precipitated with ammonium sulfate. This protein pellet was dialyzed and used for CapG purification...
March 18, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28323168/production-and-characterization-of-a-highly-pure-rna-polymerase-holoenzyme-from-mycobacterium-tuberculosis
#17
Omar Herrera-Asmat, Lucyna Lubkowska, Mikhail Kashlev, Carlos J Bustamante, Daniel G Guerra, Maria L Kireeva
Recent publications have shown that active RNA polymerase (RNAP) from Mycobacterium tuberculosis (MtbRNAP) can be produced by expressing all four subunits in a single recombinant Escherichia coli strain [1-3]. By reducing the number of plasmids and changing the codon usage of the Mtb genes in the co-expression system published by Banerjee et al. [1], we present a simplified, detailed and reproducible protocol for the purification of recombinant MtbRNAP containing the ω subunit. Moreover, we describe the formation of ternary elongation complexes (TECs) with a short fluorescence-labeled RNA primer and DNA oligonucleotides, suitable for transcription elongation studies...
March 18, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28323167/high-hydrostatic-pressure-enables-almost-100-refolding-of-recombinant-human-ciliary-neurotrophic-factor-from-inclusion-bodies-at-high-concentration
#18
Qi Wang, Yongdong Liu, Chun Zhang, Fangxia Guo, Cui Feng, Xiunan Li, Hong Shi, Zhiguo Su
Protein refolding from inclusion bodies (IBs) often encounters a problem of low recovery at high protein concentration. In this study, we demonstrated that high hydrostatic pressure (HHP) could simultaneously achieve high refolding concentration and high refolding yield for IBs of recombinant human ciliary neurotrophic factor (rhCNTF), a potential therapeutic for neurodegenerative diseases. The use of dilution refolding obtained 18% recovery at 3 mg/mL, even in the presence of 4 M urea. In contrast, HHP refolding could efficiently increase the recovery up to almost 100% even at 4 mg/mL...
March 18, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28315746/optimized-protocol-for-soluble-prokaryotic-expression-purification-and-structural-analysis-of-human-placenta-specific-1-plac1
#19
Mahboobeh Nazari, Amir-Hassan Zarnani, Roya Ghods, Rahman Emamzadeh, Somayeh Najafzadeh, Arash Minai-Tehrani, Jafar Mahmoudian, Maryam Yousefi, Sedigheh Vafaei, Sam Massahi, Mohammad-Reza Nejadmoghaddam
Placenta specific -1 (PLAC1) has been recently introduced as a small membrane-associated protein mainly involved in placental development. Expression of PLAC1 transcript has been documented in almost one hundred cancer cell lines standing for fourteen distinct cancer types. The presence of two disulfide bridges makes difficult to produce functional recombinant PLAC1 in soluble form with high yield. This limitation also complicates the structural studies of PLAC1, which is important for prediction of its physiological roles...
March 16, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28315745/production-of-low-expressing-recombinant-cationic-biopolymers-with-high-purity
#20
Xuguang Chen, Alireza Nomani, Niket Patel, Arash Hatefi
The growing complexity of recombinant biopolymers for delivery of bioactive agents requires the ability to control the biomaterial structure with high degree of precision. Genetic engineering techniques have provided this opportunity to synthesize biomaterials in an organism such as E. coli with full control over their lengths and sequences. One class of such biopolymers is recombinant cationic biopolymers with applications in gene delivery, regenerative medicine and variety of other biomedical applications...
March 16, 2017: Protein Expression and Purification
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