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Protein Expression and Purification

Wenjun Zhu, Tao Hong, Weikai Wang, Yuchen Wei, Xueling Mao, Xiaoting Qiu
Carboxyl-terminal repeat domain (CTD) of the largest subunit Rpb1 of RNA polymerace II is essential for transcription regulation. Heptapeptide repeat of CTD of Rpb1 is phosphorylated by carboxyl-terminal repeat domain kinase (CTDK-I), composed of CTK1, CTK2 and CTK3, in order to regulate transcription and transcription associated processes. The yeast specific protein CTK3 binds to cyclin CTK2 to form a heterodimer serving as a regulational factor to control CTK1 activity by binding to CTK1. Structural information of CTK2-CTK3 complex is yet to be elucidated...
September 18, 2018: Protein Expression and Purification
Julieta Cerezo, María Emilia Smith, Julián Rodríguez Talou
This work describes a novel strategy for the integrated expression and purification of recombinant proteins in Pichia pastoris cultures. Hydrophobins can be used as fusion tags, proteins fused to them alter their hydrophobicity and can be purified by aqueous two-phase systems (ATPS) based on non-ionic surfactants. Here, the consensus dengue virus envelope protein domain III fused to hydrophobin I of Trichoderma reesei was expressed in Pichia pastoris cultures and an in situ product removal by an ATPS using a non-ionic detergent, (Triton X-114) was performed...
September 18, 2018: Protein Expression and Purification
Gheorghita Menghiu, Vasile Ostafe, Radivoje Prodanovic, Rainer Fischer, Raluca Ostafe
Chitin is an abundant biopolymer found mainly in the exoskeleton of crustaceans and insects. The degradation of chitin using chitinases is one way to address the accumulation of chitin waste streams in the environment, and research has therefore focused on the identification, improvement and expression of suitable enzymes. Here we describe the production, purification and characterization of Bacillus licheniformis chitinase A in the Pichia pastoris expression system. Optimal enzyme activity occurred at pH 4...
September 17, 2018: Protein Expression and Purification
Giancarlo Abis, Rebecca L Charles, Philip Eaton, Maria R Conte
The human soluble Epoxide Hydrolase (hsEH) is an enzyme involved in the hydrolysis of endogenous anti-inflammatory and cardio-protective signalling mediators known as epoxyeicosatrienoic acids (EETs). EETs' conversion into the corresponding diols by hsEH generates non-bioactive molecules, thereby the enzyme inhibition would be expected to enhance the EETs bioavailability, and their beneficial properties. Numerous inhibitors have been developed to target the enzyme, some of which are showing promising antihypertensive and anti-inflammatory properties in vivo...
September 12, 2018: Protein Expression and Purification
Emmanuella E Fletcher, Dandan Yan, Anthony A Kosiba, Yang Zhou, Haifeng Shi
Proteins are essential throughout the biological and biomedical sciences and the purification strategies of proteins of interest have advanced over centuries. Elastin-like polypeptides (ELPs) are compound polymers that have recently been highlighted for their sharp and reversible phase transition property when heated above their lower critical solution temperature (LCST). ELPs preserve this behavior when fused to a protein, and as a result providing a simple method to isolate a recombinant ELP fusion protein from cell contaminants by taking the solution through the soluble and insoluble phase of the ELP fusion protein, a technique designated as the inverse transition cycle (ITC)...
September 11, 2018: Protein Expression and Purification
Nihar Nalini Mohanty, Sathish Bhadravathi Shivachandra, Sanchay Kumar Biswas, Vijay Nagaraj, J B Thaslim, D Narendra Babu, R Yogisharadhya, Divakar Hemadri
Strategic design and suitable purification techniques are of paramount importance in the production of recombinant proteins, if intended for use in a diagnostic assay. However, there is no single protocol that can be universally adopted for obtaining proteins in requisite quality and quantity across various platforms. In this study, we have targeted proteins of bluetongue virus (BTV), which is the causative agent of an arthropod-borne infectious disease in ruminants. Traditionally, serological diagnosis of the disease has rested upon either virus neutralization test or on an ELISA test that employed a recombinant structural (VP1, VP7) protein...
September 11, 2018: Protein Expression and Purification
Herlinda Clement, Gerardo Corzo, Edgar Neri-Castro, Ivan Arenas, Silvia Hajos, Adolfo R de Roodt, Elba Villegas
A mRNA transcript that codes for a phospholipase (PLA2 ) was isolated from a single venom gland of the Bothrops ammodytoides viper. The PLA2 transcript was cloned onto a pCR® 2.1-TOPO vector and subsequently expressed heterologously in the E. coli strain M15, using the pQE30 vector. The recombinant phospholipase was named rBamPLA2_1, and is composed of an N-terminal fusion protein of 16 residues, along with 122 residues from the mature protein that includes 14 cysteines that form 7 disulfide bonds. Following bacterial expression, rBamPLA2_1 was obtained from inclusion bodies and extracted using a chaotropic agent...
September 8, 2018: Protein Expression and Purification
Yifeng Li
Caprylic acid (CA), a naturally occurring eight-carbon fatty acid, has long been used as albumin stabilizer, non-IgG fraction precipitant and bactericidal agent in pharmaceutical industry. The mechanisms through which CA achieves its effects have been correlated with the molecule's protein/lipid binding capacity conferred by its octyl moiety. This article, following an initial review of CA's historical applications, introduces CA's relatively new application in downstream processing of monoclonal antibodies (mAbs)...
September 8, 2018: Protein Expression and Purification
Xiaoyue Sun, Wei Shen, Yanyun Gao, Menghao Cai, Mian Zhou, Yuanxing Zhang
Alginate lyase digestion is an efficient way to degrade alginate into oligosaccharides, which are useful in various areas including chemistry, pharmacy and food industry. Here we determined the sequence of Vibrio sp. QY102 sourced alginate lyase, and set up its heterologous expression in E. coli. We improved its secretion efficiency by replacing the original secretive sequence by E. coli specific signal peptide ompA. We successfully purified the full-length protein in shake flask culture, however, degradation happened during fed batch cultivation...
September 8, 2018: Protein Expression and Purification
Yang Zong, Xiao Tan, Jingjing Xiao, Xinyu Zhang, Xiaoli Xia, Huaichang Sun
Recombinant interferon-α (rIFN-α) has been widely used for treating viral infections. However, the clinical efficacy of unmodified rIFN-α is limited due to small molecular size and rapid clearance from circulation. In this study we developed a novel strategy for half-life extension of porcine IFN-α (PoIFN-α) by fusion to the immunoglobulin (Ig)-binding C2 domain of streptococcal protein G (SPG). The coding sequences for PoIFN-α6 and SPG C2 domain, with a tobacco etch virus (TEV) protease recognition sequence introduced at the 5-end, were cloned into an elastin-like polypeptide (ELP) fusion expression vector and expressed as an ELP-PoIFNα-C2 fusion protein...
August 27, 2018: Protein Expression and Purification
Fabiane Cristina Dos Santos, Ione Parra Barbosa-Tessmann
Some microorganisms can produce cyclodextrin glycosyltransferases, which degrades starch by catalyzing cyclization and giving rise to cyclodextrin. Thus, to fully degrade starch, microorganisms can also synthesize cyclodextrinases, which hydrolyze cyclodextrins forming linear α(1,4)-linkage containing oligosaccharides, that can be degraded by other enzymes. In this work, a truncated gene, without the signal peptide sequence, encoding the cyclodextrinase HMPREF9710_01718 from Massilia timonae was PCR amplified, cloned, and expressed in E...
August 24, 2018: Protein Expression and Purification
Xudong Zhang, Tao Chen, Yifeng Li
Aggregation is a major concern for therapeutic monoclonal antibody (mAb), as aggregates reduce drug efficacy and safety. In addition to aggregation control, aggregate removal by downstream processing is crucial. Hydrophobic and mixed-mode resins are widely used for aggregate removal in different cases, but they are seldom compared side by side. In this study, the aggregate removing capability of eight resins belonging to different chromatographic types was demonstrated by a case study. This work, by providing multiple options for aggregate removal, allows more flexibility to be gained in downstream processing...
August 23, 2018: Protein Expression and Purification
Priyanka Priyanka, Gemma Kinsella, Gary T Henehan, Barry J Ryan
The Pseudomonas sp. have been long recognized for their exogenous lipolytic activities yet the genus still contains a lot of unexplored strains. Due to the versatile metabolic machinery and their potential for adaptation to fluctuating environmental conditions Pseudomonas sp. are of great interest for biotechnological applications. In this study, a new extracellularly produced lipolytic enzyme from Pseudomonas sp. (P. reinekei) was purified and characterized. The production of lipase from P. reinekei (H1) was enhanced 10-fold by optimizing the nitrogen source...
August 22, 2018: Protein Expression and Purification
Ajay B Maghodia, Christoph Geisler, Donald L Jarvis
No abstract text is available yet for this article.
August 21, 2018: Protein Expression and Purification
Akash Pandhare, Antonia G Stuebler, Elham Pirayesh, Michaela Jansen
The main principles of higher-order protein oligomerization are elucidated by many structural and biophysical studies. An astonishing number of proteins self-associate to form dimers or higher-order quaternary structures which further interact with other biomolecules to elicit complex cellular responses. In this study, we describe a simple and convenient approach to determine the oligomeric state of purified protein complexes that combines implementation of a novel form of clear-native gel electrophoresis and size exclusion chromatography in line with multi-angle light scattering...
August 18, 2018: Protein Expression and Purification
Dominik Georg Sauer, Magdalena Mosor, Anna-Carina Frank, Florian Weiß, Anna Christler, Nicole Walch, Alois Jungbauer, Astrid Dürauer
A two-step purification process for human basic fibroblast growth factor (FGF-2) from clarified E. coli homogenate has been developed in which the impurity level after the second step is below the limit of quantification. Endotoxin content is cleared to 0.02 EU/μg FGF-2 and the overall yield is 67%. The performance of the cation exchanger Carboxymethyl-Sepharose Fast Flow (CM-SFF) was compared to the affinity resin Heparin-SFF regarding the impurity profile and product quality in the elution peak. The CM-SFF eluate was further purified using hydrophobic interaction resin Toyopearl-Hexyl-650C...
August 18, 2018: Protein Expression and Purification
Matías Sebastián Lorch, María Soledad Collado, Marcelo Horacio Argüelles, Rosana Paola Rota, Lorena Ivana Spinsanti, Mario Enrique Lozano, Sandra Elizabeth Goñi
Saint Louis encephalitis virus (SLEV) and West Nile virus (WNV) are two of the major causes of arboviral encephalitis in the Americas. The co-circulation of related flaviviruses in the Americas and prior vaccination against flaviviruses pose problems to the diagnostic specificity of serological assays due to the development of cross-reactive antibodies. An accurate diagnosis method capable of differentiating these related viruses is needed. NS1 is a glycosylated, nonstructural protein, of about 46 kDa which has a highly conserved structure...
August 17, 2018: Protein Expression and Purification
Zhaokui Du, Junmin Li
Kandelia candel, a major species of mangrove in the tropical and subtropical area, is susceptible to low temperature in winter. K. candel was introduced into Zhejiang Province (the northern margin of South China) several decades ago, and suffered from low temperature causing growth retardation, in server cases, even death. To explore the molecular mechanisms of cold acclimation in K. candel, a novel C-repeat binding factor gene KcCBF3 (Genbank accession no. KF111715) of 729 bp open reading frame (ORF) encoding a protein of 242 amino acid residues was isolated, expressed, purified and characterized...
August 16, 2018: Protein Expression and Purification
Zheng-Gang Han, Ji-Wen Zhang, Xiao-Fang Jiang, Jiang-Ke Yang
The α-galactosidases, which can catalyze the removal of α-1,6-linked terminal galactose residues from galactooligosaccharide materials, have good potential for industrial applications. The high-level and efficient secretion of the α-galactosidases into the extracellular space has greatly simplified the downstream bioengineering process, facilitating their bioapplications. In this study, the effects of gene dosage and endoplasmic reticulum secretion-associated factors (ERSAs) on the secretory expression of an α-galactosidase gene derived from a Aspergillus oryzae strain were investigated by constructing multicopy expression cassettes and coexpressing the α-galactosidase gene with ERSAs...
August 11, 2018: Protein Expression and Purification
Wolfgang Becker, Anne Scherer, Christine Faust, David Kornblüh Bauer, Simone Scholtes, Ercole Rao, Joachim Hofmann, Rolf Schauder, Thomas Langer
The drug discovery process in the biopharmaceutical industry usually starts with the generation of plasmids coding for certain proteins. Due to advances in cloning techniques the generation of thousands of different plasmids is not a limiting factor anymore. The next step is the expression and evaluation of the proteins. In recent years significant progress has been made in the miniaturization of protein expression and purification. These processes have been adapted to robotic platforms and hundreds of proteins can be expressed and purified in parallel...
August 10, 2018: Protein Expression and Purification
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