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Protein Expression and Purification

Christoph Geisler, Donald L Jarvis
Insect cells are widely used for recombinant protein expression, typically as hosts for recombinant baculovirus vectors, but also for plasmid-mediated transient transfection or stable genetic transformation. Insect cells are used to express proteins for research, as well as to manufacture biologicals for human and veterinary medicine. Recently, several insect cell lines used for recombinant protein expression were found to be persistently infected with adventitious viruses. This has raised questions about how these infections might affect research performed using those cell lines...
November 10, 2017: Protein Expression and Purification
Murad Mollaev, Neonila Gorokhovets, Elena Nikolskaya, Maria Faustova, Arthur Zabolotsky, Maria Sokol, Oksana Tereshenko, Olga Zhunina, Vitaliy Shvets, Evgeniy Severin, Nikita Yabbarov
A wide range of methods are known to increase the prokaryotic intracellular recombinant proteins solubility, for instance, growth at low temperature, supplementation of culture media with "chemical chaperones" (proline, glycine-betaine, and trehalose), co-expression with chaperones or highly soluble fusion partners. As an alternative, we have introduced the polyglutamate tag, which, as it has been shown, increased the protein solubility and facilitated folding. In this study we evaluated the minimal quantity of high density negatively charged EEEEVE amino acid repeats (pGlu) necessary to switch the recombinant receptor-binding domain of human alpha-fetoprotein (rbdAFP) expression almost entirely from the inclusion bodies to the soluble cytoplasmic fraction in E...
November 7, 2017: Protein Expression and Purification
S De Waele, I Vandenberghe, B Laukens, S Planckaert, S Verweire, I N A Van Bogaert, W Soetaert, B Devreese, K Ciesielska
The Starmerella bombicola lactone esterase (SBLE) is a novel enzyme that, in vivo, catalyzes the intramolecular esterification (lactonization) of acidic sophorolipids in an aqueous environment. In fact, this is an unusual reaction given the unfavorable conditions for dehydration. This characteristic strongly contributes to the potential of SBLE to become a 'green' tool in industrial applications. Indeed, lactonization occurs normally in organic solvents, an application for which microbial lipases are increasingly used as biocatalysts...
November 4, 2017: Protein Expression and Purification
Dimitrius Santiago Guimarães, Marcelo Damário Gomes
Autophagy is the process of degradation of intracellular proteins through the lysosome. Members of the tripartite motif (TRIM) proteins have shown to directly recognize autophagic cargo and also to act as a hub for the phagophore nucleation complex. The TRIM proteins are classically characterized by the presence of an amino-terminal RING domain and a B-box domain followed by a coiled coil domain. Although regarded as ubiquitin E3 ligases, this activity has been shown only for a minor set of the 79 human TRIM proteins...
November 4, 2017: Protein Expression and Purification
Youyou Lu, Nana Wang, Jiao He, Yanfang Li, Xiaoning Gao, Lili Huang, Xia Yan
Saccharothrix yanglingensis Hhs.015, a new type of rare actinomycete, was isolated from the roots of cucumber. A novel chitinase gene was cloned from S. yanglingensis Hhs.015 and overexpressed as a soluble protein Chi6769 (77.9 kDa) in Escherichia coli. Chi6769 was purified by HisTrap HP affinity chromatography with optimal pH of 7.0. The enzymatic hydrolysis assay revealed that Chi6769 was capable of hydrolyzing chitin to (GlcNAc)3, (GlcNAc)2 and GlcNAc. (GlcNAc)2 was the main hydrolyzate. The antifungal activity result showed that Chi6769 exhibited strong antifungal activity toward Valsa mali 03-8...
November 1, 2017: Protein Expression and Purification
Seyede Zohreh Mirahmadi-Zare, Fatemeh Aboutalebi, Maryam Allafchian, Leila Pirjamali, Mohammad-Hossein Nasr-Esfahani
Magnetic nanoparticles NiFe2O4 was synthesized and covered in the silicate lattice of (3-Aminopropyl) triethoxysilane (APS) by the sol-gel process. Subsequently, the EDTA-dianhydride was attached to the amino surface of magnetic nanoparticles (MNPs) during the nucleophilic attack. This polycarboxylic layer trapped the high level of nickel ions for selective bonding to the His-tagged recombinant protein. The surface of MNPs was investigated by TEM, XRD, SEM (EDSA), VSM, BET, FT-IR and zeta potential analysis which characterized the size, chemical lattice, morphology, magnetic strength, specific surface area, functional groups and charge of the surface of nanoparticles...
October 28, 2017: Protein Expression and Purification
R Sindhu, H K Manonmani
l-asparaginase (E.C., an anti-cancer drug has been used in the treatment of acute lymphoblastic leukemia. A novel source of l-asparaginase from Pseudomonas fluorescens was addressed in the present studies. The ANS gene in Pseudomonas fluorescens MTCC 8127 which produces l-asparaginase was cloned and expressed in E. coli BL21 (DE3). The expressed recombinant protein (PfAns) which was purified, showed l-asparaginase activity. The enzyme was further characterized. The pH and temperature optima were found to be 7...
October 25, 2017: Protein Expression and Purification
Sarah M Plucinsky, Kyle T Root, Kerney Jebrell Glover
The purification of membrane proteins can be challenging due to their low solubility in conventional detergents and/or chaotropic solutions. The introduction of fusion systems that promote the formation of inclusion bodies has facilitated the over-expression of membrane proteins. In this protocol, we describe the use of perfluorooctanoic acid (PFOA) as an aid in the purification of highly hydrophobic membrane proteins expressed as inclusion bodies. The advantage of utilizing PFOA is threefold: first, PFOA is able to reliably solubilize inclusion bodies, second, PFOA is compatible with nickel affinity chromatography, and third, PFOA can be efficiently dialyzed away to produce a detergent free sample...
October 21, 2017: Protein Expression and Purification
Darya O Ashcheulova, Lina V Efimova, Aliaksandr Ya Lushchyk, Aliaksei V Yantsevich, Alexander N Baikov, Alexandra G Pershina
Radiolabeled peptides derived from ubiquicidine (UBI) are of great interest for early and highly accurate scintigraphic detection and differentiation of infection and sterile inflammation. In the present work the recombinant antimicrobial peptide UBI18-35 - a fragment of the human natural cationic peptide ubiquicidine - was produced in Escherichia coli for the first time. The insoluble expression of the peptide in fusion with ketosteroid isomerase provided high yield, about 6 mg of UBI18-35 per liter. We developed an approach to produce the antimicrobial peptide UBI18-35, that encompasses inclusion body isolation and size exclusion chromatography...
October 21, 2017: Protein Expression and Purification
Weiwei Zheng, Liu Yang, Chenggu Cai, Jinfeng Ni, Bo Liu
The sweet protein monellin has high sweet potency with limited stability. In this study, 3 double-sites mutants (E2N/E23A, E2N/Y65R and E23A/Y65R) of the single-chain monellin (MNEI) were constructed. The proteins were expressed in E. coli BL21 and purified to homogeneity by nickel affinity chromatography with yields above 10 mg/L cell culture. Introduction of a sweeter mutant E2N into E23A or Y65R (E2N/E23A and E2N/Y65R) led to about 3-fold increase of sweetness, while addition of a more stable mutant E23A into E2N or Y65R (E2N/E23A and E23A/Y65R) resulted in improved thermal stability (about 10 °C)...
October 16, 2017: Protein Expression and Purification
Xuan Tang, Shuangshuang Wu, Xiaofeng Wang, Qing Gu, Ping Li
Strains belonging to the genus of Staphylococci, such as Staphylococcus aureus are common pathologic bacteria which may cause nosocomial cross infection and food contamination. Plantaricin EF (PlnEF), a two-peptide nonlantibiotic bacteriocin produced by Lactobacillus plantarum strains, shows great inhibitory effects against Gram-positive Staphylococci strains. To overproduce this two-peptide bacteriocin, plnE and plnF genes were cloned into pET32a (+) vector and heterologously expressed in Escherichia coli by fusion with His6-tag in this study...
October 14, 2017: Protein Expression and Purification
Keding Cheng, Angela Sloan, Brooks Waitt, Robert Vendramelli, Debra Godal, Sharon L R Simon, Joe O'Neil, Michael Carpenter, Dave Jackson, Jane Eastlake, Gary Mallinson, J David Knox
BACKGROUND: Bacterially-produced recombinant prion protein (rPrP) has traditionally been used for in vitro fibrillation assays and reagent development for prion disease research. In recent years, it has also been used as a substrate for real-time quaking-induced conversion (RT-QuIC), a very sensitive method of detecting the presence of the misfolded, disease-associated isoform of the prion protein (PrP(d)). Multi-centre trials have demonstrated that RT-QuIC is a suitably reliable and robust technique for clinical practice; however, in the absence of a commercial supplier of rPrP as a substrate for RT-QuIC, laboratories have been required to independently generate this key component of the assay...
October 12, 2017: Protein Expression and Purification
R Vijayakumar, Timir Tripathi
We report the molecular cloning, expression, and single-step homogeneous purification of a full-length asparaginyl tRNA synthetase (NRS) from Fasciola gigantica (FgNRS). Fasciola gigantica is a parasitic liver fluke of the class Trematoda. It causes fascioliasis that infects the liver of various mammals, including humans. Aminoacyl tRNA synthetases (AARS) catalyze the first step of protein synthesis. They attach an amino acid to its cognate tRNA, forming an amino acid-tRNA complex. The gene that codes for FgNRS was generated by amplification by polymerase chain reaction...
October 12, 2017: Protein Expression and Purification
Beatriz Nogueira Messias Miranda, Wesley Luzetti Fotoran, Fernanda Canduri, Dulce Helena Ferreira Souza, Gerhard Wunderlich, Emanuel Carrilho
The role of Alpha folate receptors (FRα) in folate metabolism and cancer development has been extensively studied. The reason for this is not only associated to its direct relation to disease development but also to its potential use as a highly sensitive and specific biomarker for cancers therapies. Over the recent years, the crystal structures of human FRα complexed with different ligands were described relying on an expensive and time-consuming production process. Here, we constructed an efficient system for the expression and purification of a human FRα in E...
October 5, 2017: Protein Expression and Purification
Hongbo Li, Yuxian Xia
Scorpion long-chain insect neurotoxins have important potential application value in agricultural pest control. The difficulty of obtaining natural toxins is the major obstacle preventing analyses of their insecticidal activity against more agricultural insect pests. Here we cloned the insect neurotoxin BjαIT gene into the pET32 expression vector and expressed the resulting thioredoxin (Trx)-BjαIT fusion protein in Escherichia coli. Soluble Trx-BjαIT was expressed at a high level when induced at 18 °C with 0...
October 4, 2017: Protein Expression and Purification
Hye-Rim Lee, Jin Choi, Seung Hoon Lee, Mi-La Cho, Dae-Myung Jue
A20 (also known as TNFAIP3) is a potent anti-inflammatory protein that suppresses many intracellular signaling pathways induced by inflammatory cytokines and bacterial and viral pathogens. The anti-inflammatory function of A20 depends on its modulation of or binding to polyubiquitin chains on key signaling proteins in the nuclear factor-κB (NF-κB) pathway. To test whether A20 can be used as therapeutic agent in these inflammatory diseases, we prepared a recombinant cell-penetrating form of A20 (TAT-A20) for intracellular delivery and examined its effect on tumor necrosis factor-α (TNFα)-induced NF-κB activation...
October 4, 2017: Protein Expression and Purification
Yueting Zheng, Qitao Liu, Huanhuan Shen, Guoyu Yang
Reversible N(ε)-lysine (N(ε)-Lys) acetylation is a dynamic post-translational modification. Genetic incorporation of N(ε)-acetyllysine (N(ε)-AcK) into the specific site of a protein is a powerful method for producing recombinant protein with acetylation and studying the functional role of protein acetylation. Because of the universal existence of deacetylase such as CobB in vivo, the acetyl group of N(ε)-AcK may be removed from recombinant protein. So in the process of incorporating acetyl lysine into protein, nicotinamide (NAM), a lysine deacetylase (KDAC) inhibitor, is needed to inhibit the KDAC activity and protect the acetyl group of N(ε)-acetyllysine incorporated from removal in vivo...
October 3, 2017: Protein Expression and Purification
Kevin E Acero-Navarro, Mariella Jiménez-Ramírez, Miguel A Villalobos, Rocío Vargas-Martínez, Hugo V Perales-Vela, Roberto Velasco-García
Glucose-6-phosphate dehydrogenase (G6PDH) (EC plays an important role in the human pathogen Pseudomonas aeruginosa because it generates NADPH, an essential cofactor for several biosynthetic pathways and antioxidant enzymes. P. aeruginosa G6PDH is also a key enzyme in the metabolism of various carbon sources, such as glucose, glycerol, fructose, and mannitol. Understanding the kinetic characteristics and mechanisms that control the activity of this enzyme is crucial for future studies in this context...
October 3, 2017: Protein Expression and Purification
Ke An, Xiaochong Shi, Fangyuan Cui, Jingguang Cheng, Na Liu, Xia Zhao, Xiao-Hua Zhang
Agar, usually extracted from seaweed, has a wide variety of industrial applications due to its gelling and stabilizing characteristics. Agarases are the enzymes which hydrolyze agar into agar oligosaccharides. The produced agar oligosaccharides have been widely used in cosmetic, food, and medical fields due to their biological functions. A beta-agarase gene, YM01-1, was cloned and expressed from a marine bacterium Catenovulum agarivorans YM01(T). The encoding agarase of YM01-1 consisted of 331 amino acids with an apparent molecular mass of 37...
October 3, 2017: Protein Expression and Purification
Yunnuo Shi, Scott A Halperin, Song F Lee
Delivering antigen via molecules specifically targeting receptors on the surface of antigen-presenting cells is a strategy to improve immune responses. In this study, an antigen-targeting fusion protein (OVA-CD40LS) composed of the C-terminal fragment of ovalbumin and the extracellular domain of mouse CD40 ligand was constructed by genetic fusion. The OVA-CD40LS and the control OVA (rOVA) genes were cloned in Escherichia coli and over-expressed as insoluble proteins. The rOVA protein was purified from the insoluble fraction of E...
September 30, 2017: Protein Expression and Purification
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