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Affinity chromatography

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https://www.readbyqxmd.com/read/28448741/chromophore-attachment-to-fusion-protein-of-streptavidin-and-recombinant-allophycocyanin-alpha-subunit
#1
Jing Wu, Huaxin Chen, Peng Jiang
The fusion protein (SLA) of streptavidin and allophycocyanin alpha subunit (holo-ApcA) was biosynthesized in Escherichia coli by a dual plasmid system. The recombinant SLA, purified by affinity chromatography, showed spectral properties similar to natural allophycocyanin alpha subunit (ApcA). Spectral and Zinc staining analysis indicated that the recombinant SLA covalently bound phycocyanobilin (PCB). To improve chromophorylation rate of recombinant SLA, an in vitro chromophore attachment reaction system was established, which contained partially chromophylated SLA, PCB and lyase CpcS...
April 27, 2017: Bioengineered
https://www.readbyqxmd.com/read/28445763/myosin-rod-hypophosphorylation-and-cb-kinetics-in-papillary-muscles-from-a-tnc-a8v-ki-mouse-model
#2
Masataka Kawai, Jamie R Johnston, Tarek Karam, Li Wang, Rakesh K Singh, Jose R Pinto
The cardiac troponin C (TnC)-A8V mutation is associated with hypertrophic and restrictive cardiomyopathy (HCM and RCM) in human and mice. The residue affected lies in the N-helix, a region known to affect Ca(2+)-binding affinity to the N-terminal domain. Here we report on the functional effects of this mutation in skinned papillary muscle fibers from homozygous knock-in TnC-A8V mice. Muscle fibers from left ventricle were activated at 25°C under the ionic conditions of working cardiomyocytes. The pCa-tension relationship showed a 3× increase in Ca(2+)-sensitivity and a decrease (0...
April 25, 2017: Biophysical Journal
https://www.readbyqxmd.com/read/28445001/inhibition-properties-of-some-flavonoids-on-carbonic-anhydrase-i-and-ii-isoenzymes-purified-from-human-erythrocytes
#3
Zübeyir Huyut, Şükrü Beydemir, İlhami Gülçin
Carbonic anhydrases (CAs, E.C.4.2.1.1) play a critical role in many important physiological events and treatment of some diseases. Flavonoids or phenolic compounds have been discovered as novel CAs inhibitors instead of the traditional sulfonamides, with different binding to CAs, pro-drug activities, and new inhibition mechanisms. Here, we investigated the inhibition effects of some flavonoids including malvin, callistephin, oenin, pelargonin, silychristin, and 1-(4-methoxyphenyl)-2-methyl-3-nitro-1-H-indol-6-ol (ID-8) against hCA I and II, which purified from human erythrocytes by affinity column chromatography...
April 26, 2017: Journal of Biochemical and Molecular Toxicology
https://www.readbyqxmd.com/read/28441606/growth-inhibition-and-antibiofilm-potential-of-ag-nanoparticles-coated-with-lectin-an-arthropod-immune-molecule
#4
Sangily Jayanthi, Sathappan Shanthi, Baskaralingam Vaseeharan, Narayanan Gopi, Marimuthu Govindarajan, Naiyf S Alharbi, Shine Kadaikunnan, Jamal M Khaled, Giovanni Benelli
Lectins from the haemolymph of arthropods, including crustaceans, are molecules potentially involved in the immune recognition and phagocytosis. Here, lectin was purified from the haemolymph of blue swimmer crab Portunus pelagicus, using mannose-coupled sepharose CL-4B affinity column chromatography. In SDS-PAGE analysis, lectin showed a molecular mass of approximately 155kDa. The synthesis of lectin-coated silver nanoparticles (lectin-coated AgNPs) was confirmed by UV-Vis spectroscopy, XRD, FTIR, SEM, TEM, SAED, and EDX analysis...
April 13, 2017: Journal of Photochemistry and Photobiology. B, Biology
https://www.readbyqxmd.com/read/28439850/heterologous-expression-of-a-streptomyces-cyaneus-laccase-for-biomass-modification-applications
#5
Selin Ece, Camilla Lambertz, Rainer Fischer, Ulrich Commandeur
Laccases are used for the conversion of biomass into fermentable sugars but it is difficult to produce high yields of active laccases in heterologous expression systems. We overcame this challenge by expressing Streptomyces cyaneus CECT 3335 laccase in Escherichia coli (ScLac) and we achieved a yield of up to 104 mg L(-1) following purification by one-step affinity chromatography. Stability and activity assays using simple lignin model substrates showed that the purified enzyme preparation was active over a broad pH range and at high temperatures, suggesting it would be suitable for biomass degradation...
December 2017: AMB Express
https://www.readbyqxmd.com/read/28439623/identification-of-multiple-sources-of-the-acidic-charge-variants-in-an-igg1-monoclonal-antibody
#6
Shiwei Miao, Panpan Xie, Mao Zou, Li Fan, Xuping Liu, Yan Zhou, Liang Zhao, Ding Ding, Haibin Wang, Wen-Song Tan
Charge variants, especially acidic charge variants, of recombinant monoclonal antibodies are the major critical quality attributes in the biotechnology industry due to their potential influence on stability and biological activity. The chemical properties of the acidic charge variants have been challenging to fully characterize, and it is critical for process development and optimization. To completely understand the multiple sources of acidic charge variants, the major charge forms of an IgG1 monoclonal antibody were firstly isolated and then analyzed by a battery of characterization tools...
April 24, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28436221/the-iron-chaperone-protein-cyay-from-vibrio-cholerae-is-a-heme-binding-protein
#7
Takeshi Uchida, Noriyuki Kobayashi, Souichiro Muneta, Koichiro Ishimori
CyaY is an iron transport protein for Fe-S cluster biosynthetic systems and a ferrochelatase that catalyzes insertion of Fe(2+) into protoporphyrin IX. Here, we found that CyaY has the ability to bind heme as well as iron, exhibiting a dissociation constant for heme of 21 ± 6 nM. Absorption and resonance Raman spectra revealed that both ferric and ferrous forms of heme were bound to an anionic ligand (e.g., tyrosine and/or cysteine). Consistent with this, mutagenesis studies showed that Tyr67 and Cys78 are possible heme ligands of CyaY...
April 24, 2017: Biochemistry
https://www.readbyqxmd.com/read/28433055/pyrenyl-carbon-nanostructures-for-ultrasensitive-measurements-of-formaldehyde-in-urine
#8
Gayan Premaratne, Sabrina Farias, Sadagopan Krishnan
Measurement of ultra-low (e.g., parts-per-billion) levels of small-molecule markers in body fluids (e.g., serum, urine, saliva) involves a considerable challenge in view of designing assay strategies with sensitivity and selectivity. Herein we report for the first time an amperometric nano-bioelectrode design that uniquely combines 1-pyrenebutyric acid units pi-pi stacked with carboxylated multiwalled carbon nanotubes on the surface of gold screen printed electrodes for covalent attachment of NAD(+) dependent formaldehyde dehydrogenase (FDH)...
June 1, 2017: Analytica Chimica Acta
https://www.readbyqxmd.com/read/28432819/identification-of-bioactive-compounds-in-shaoyao-gancao-decoction-using-%C3%AE-2-adrenoceptor-affinity-chromatography
#9
Zehua Li, Haiyang Gao, Jiangying Li, Yajun Zhang
Shaoyao-Gancao decoction, a Chinese herbal formula, is composed of Paeoniae Radix alba and Glycyrrhiza Radix et rhizoma. It has been widely used to treat muscle spasms and asthma. However, little is known about the bioactive components of Shaoyao-Gancao decoction. In the present study, the bioactive compounds in water-extract of Shaoyao-Gancao decoction were separated by the immobilized β2 -adrenoceptor affinity column and identified using quadrupole time-of-flight mass spectrometry. The affinity constants of the separated compounds that bound to β2 -adrenoceptor were determined by frontal analysis...
April 22, 2017: Journal of Separation Science
https://www.readbyqxmd.com/read/28432772/studies-on-the-chitin-chitosan-binding-properties-of-six-cuticular-proteins-analogous-to-peritrophin-3-from-bombyx-mori
#10
M Qu, Y Ren, Y Liu, Q Yang
Chitin deacetylation is required to make the cuticle rigid and compact through chitin chain crosslinking. Thus it is presumed that specialized proteins are required to bind deacetylated chitin chains together. However, deacetylated-chitin binding proteins have not ever been reported. In a previous work, six cuticular proteins analogous to peritrophin 3 (CPAP3s) were found to be abundant in the moulting fluid of Bombyx mori. In this study, these BmCPAP3s (BmCPAP3-A1, BmCPAP3-A2, BmCPAP3-B, BmCPAP3-C, BmCPAP3-D1 and BmCPAP3-D2) were cloned and expressed in Escherichia coli and purified using metal-chelating affinity chromatography...
April 22, 2017: Insect Molecular Biology
https://www.readbyqxmd.com/read/28431943/expression-and-purification-of-p70%C3%AE-ct104-s6-k-a-72kda-c-terminal-truncated-p70s6-kinase-gst-fusion-protein-in-bacterial-expression-system
#11
Younis Mohammad Hazari, Irfana Reshi, Mudasir Habib, Khalid Majid Fazili
The p70ΔCT104 S6K is a 421 amino acid residue long truncated form of p70S6 kinase, with 104 amino acids residues cleaved from the carboxyl terminal end of the original protein. The p70ΔCT104 S6K was cloned in E. coli DH5α and successfully expressed in E. coli BL21 (DE3) strain. Western blot with rabbit polyclonal anti-GST antibody was used to follow the protein during expression and purification. The protein purification was achieved by affinity chromatography using Glutathione resin-agarose beads, followed by chromatography on a spin concentration column...
April 19, 2017: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/28428153/development-of-polyol-responsive-antibody-mimetics-for-single-step-protein-purification
#12
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/28426733/mini-g-proteins-novel-tools-for-studying-gpcrs-in-their-active-conformation
#13
Rony Nehmé, Byron Carpenter, Ankita Singhal, Annette Strege, Patricia C Edwards, Courtney F White, Haijuan Du, Reinhard Grisshammer, Christopher G Tate
Mini-G proteins are the engineered GTPase domains of Gα subunits. They couple to GPCRs and recapitulate the increase in agonist affinity observed upon coupling of a native heterotrimeric G protein. Given the small size and stability of mini-G proteins, and their ease of expression and purification, they are ideal for biophysical studies of GPCRs in their fully active state. The first mini-G protein developed was mini-Gs. Here we extend the family of mini-G proteins to include mini-Golf, mini-Gi1, mini-Go1 and the chimeras mini-Gs/q and mini-Gs/i...
2017: PloS One
https://www.readbyqxmd.com/read/28426042/antibodies-to-h2a-and-h2b-histones-from-the-sera-of-hiv-infected-patients-catalyze-site-specific-degradation-of-these-histones
#14
Svetlana V Baranova, Pavel S Dmitrienok, Nikita V Ivanisenko, Valentina N Buneva, Georgy A Nevinsky
Histones and their post-translational modifications have key roles in chromatin remodeling and gene transcription. Besides intranuclear functions, histones act as damage-associated molecules when they are released into the extracellular space. Administration of histones to animals leads to systemic inflammatory and toxic responses. Autoantibodies with enzymatic activities (abzymes) are distinctive features of some autoimmune and viral diseases. Electrophoretically homogeneous IgGs containing no canonical enzymes were isolated from the sera of HIV-infected patients by chromatography on several affinity sorbents including anti-histone Sepharose...
April 20, 2017: Molecular BioSystems
https://www.readbyqxmd.com/read/28421441/n-methyl-serotonin-analogues-from-the-bufo-bufo-toad-venom-interact-efficiently-with-the-%C3%AE-7-nicotinic-acetylcholine-receptors
#15
E V Kryukova, D S Lebedev, I A Ivanov, D A Ivanov, V G Starkov, V I Tsetlin, Yu N Utkin
Two low-molecular-weight compounds were isolated from the parotid gland secret of the toad Bufo bufo, which by absorption spectra and HPLC-MS/MS chromatography data correspond to di- and trimethyl derivatives of serotonin (5-hydorxytryptamine): bufotenine (confirmed by counter synthesis) and bufotenidine (5-HTQ). In experiments on competitive radioligand binding, these compounds showed a higher affinity and selectivity for neuronal α7 nicotinic acetylcholine receptors compared with the muscular cholinergic receptors...
January 2017: Doklady. Biochemistry and Biophysics
https://www.readbyqxmd.com/read/28419935/identification-of-a-host-cell-protein-impurity-in-therapeutic-protein-p1
#16
Deepti Ahluwalia, Harbhajan Dhillon, Thomas Slaney, Hangtian Song, Heather Boux, Sheetal Mehta, Lei Zhang, Anulfo Valdeza, Girija Krishnamurthy
Residual host cell proteins (HCPs) are process-related impurities present in biotherapeutics that can pose safety health risks to patients. An adequate control of HCP levels in the final product, and demonstration of HCP clearance throughout a product manufacturing process is critical for all biotherapeutic products. Developing effective downstream purification processes can be challenging as HCPs and product proteins may possess an affinity for each other or have similar physicochemical properties, resulting in co-purification...
April 9, 2017: Journal of Pharmaceutical and Biomedical Analysis
https://www.readbyqxmd.com/read/28418068/recombinant-overexpression-of-dihydroneopterin-aldolase-catalyst-potentially-regulates-folate-biofortification
#17
Dhananjay K Pandey, Atul Kumar, Jitendra S Rathore, Nagendra Singh, Bhupendra Chaudhary
We aim to investigate the prospects of increased production of folate through the overexpression of heterologous dihydroneopterin aldolase catalyst. The gene encoding aldolase catalyst was cloned into an expression vector and the induced recombinant protein was purified through metal-affinity chromatography which appeared at 14 kDa position on polyacrylamide-gel. Remarkably, a periodic increase in the extracellular and intracellular folic acid concentration was observed at 4 h growth of induced recombinant DHNA samples than control in a pH-dependent manner...
April 18, 2017: Journal of Basic Microbiology
https://www.readbyqxmd.com/read/28418008/quantitative-analyses-of-the-global-proteome-and-phosphoproteome-reveal-the-different-impacts-of-propofol-and-dexmedetomidine-on-ht22-cells
#18
Honggang Zhang, Juan Ye, Zhaomei Shi, Chen Bu, Fangping Bao
Propofol and dexmedetomidine are both commonly used anaesthetics. Although they employ two different mechanisms to induce anaesthesia, both compounds influence the hippocampus and the HT22 cell line. HT22 cells are broadly used in neurobiological research. In this study, we assessed the effects of propofol and dexmedetomidine on signalling in HT22 cells. Using the SILAC (stable isotope labelling with amino acids in cell culture) labelling technique, IMAC (immobilized metal affinity chromatography) enrichment and high-resolution LC-MS/MS (liquid chromatography tandem mass spectrometry) analysis, we investigated the quantitative proteome and phosphoproteome in HT22 cells treated with propofol or dexmedetomidine...
April 18, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28415783/mass-spectral-analysis-of-the-multikinase-inhibitor-bzg-and-its-metabolites-and-analysis-of-their-binding-to-vascular-endothelial-growth-factor-receptor-2
#19
Yan Lou, Wenqi Qiu, Zhe Wu, Qian Wang, Yunqing Qiu, Su Zeng
We previously showed that BZG is a novel multitarget kinase inhibitor, which inhibited hepatocellular carcinoma in vivo and in vitro. In the present study, we used ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) to characterize BZG and its metabolites generated in vivo. The probable metabolic mechanism was further confirmed by analysis of Phase I and Phase II metabolism in liver microsomes and with recombinant enzymes. In addition, the binding affinities of BZG metabolites to vascular endothelial growth factor receptor 2 (VEGFR2) were predicted using electronic high throughput screening (eHiTS)...
March 16, 2017: Oncotarget
https://www.readbyqxmd.com/read/28414426/two-distinct-assembly-states-of-cysteine-regulatory-complex-of-salmonella-typhimurium-are-regulated-by-enzyme-substrate-cognate-pairs
#20
Abhishek Kaushik, Mary Krishna Ekka, Sangaralingam Kumaran
Serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS), which catalyze last two steps of cysteine biosynthesis, interact and form Cysteine Regulatory Complex (CRC). The current model of Salmonella typhimurium predicts that CRC is composed of one [SAT]hexmer unit and two molecules of [OASS]dimer. However, it is not clear why [SAT]hexamer can not engage all of its six high-affinity binding sites. We examined the assembly state(s) of CRC by size exclusion chromatography (SEC), analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR) approaches...
April 17, 2017: Biochemistry
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