Artificial Cell Microcapsules Containing Genetically Engineered E. Coli dhs Cells for In-Vitro Lowering of Plasma Potassium, Phosphate, Magnesium, Sodium, Chloride, Uric Acid, Cholesterol, And Creatinine : A Preliminary Report

1999 ◽  
Vol 27 (5-6) ◽  
pp. 475-481 ◽  
Author(s):  
S. Prakash ◽  
T. M. S. Chang
Keyword(s):  
Uric Acid ◽  
Sodium Chloride ◽  
Preliminary Report ◽  
Potassium Phosphate ◽  
Plasma Potassium ◽  
Genetically Engineered ◽  
E Coli ◽  
Artificial Cell ◽  
Phosphate Magnesium

scholarly journals Prokaryotic soluble overexpression and purification of oncostatin M using a fusion approach and genetically engineered E. coli strains

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Minh Tan Nguyen ◽  
Musharrat Jahan Prima ◽  
Jung-A. Song ◽  
Julee Kim ◽  
Bich Hang Do ◽  
...  
Keyword(s):  
Metabolic Diseases ◽  
Protein A ◽  
Genetically Engineered ◽  
Oncostatin M ◽  
Glutathione S Transferase ◽  
Skeletal Tissue ◽  
E Coli ◽  
Th17 Cell Differentiation ◽  
Tissue Alteration

Abstract Human Oncostatin M (OSM), initially discovered as a tumour inhibitory factor secreted from U-937 cells, is a gp130 (IL-6/LIF) cytokine family member that exhibits pleiotropic effects in inflammation, haematopoiesis, skeletal tissue alteration, liver regeneration, cardiovascular and metabolic diseases. Cytoplasmic expression of OSM in Escherichia coli results in inclusion bodies, and complex solubilisation, refolding and purification is required to prepare bioactive protein. Herein, eight N-terminal fusion variants of OSM with hexahistidine (His6) tag and seven solubility-enhancing tags, including thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilisation substance protein A (Nusa), human protein disulphide isomerase (PDI) and the b‘a’ domain of PDI (PDIb‘a’), were tested for soluble OSM expression in E. coli. The His6-OSM plasmid was also introduced into genetically engineered Origami 2 and SHuffle strains to test expression of the protein. At 18 °C, MBP-tagged OSM was highly expressed and solubility was dramatically enhanced. In addition, His6-OSM was more highly expressed and soluble in Origami 2 and SHuffle strains than in BL21(DE3). MBP-OSM and His6-OSM were purified more than 95% with yields of 11.02 mg and 3.27 mg from a 500 mL culture. Protein identity was confirmed by mass spectroscopy, and bioactivity was demonstrated by in vitro inhibition of Th17 cell differentiation.

scholarly journals Pharmacological Basis of β-Lactamase Inhibitor Therapeutics: Tazobactam in Combination with Ceftolozane

2013 ◽  
Vol 57 (12) ◽  
pp. 5924-5930 ◽  
Author(s):  
Brian VanScoy ◽  
Rodrigo E. Mendes ◽  
Jennifer McCauley ◽  
Sujata M. Bhavnani ◽  
Catharine C. Bulik ◽  
...  
Keyword(s):  
Threshold Concentration ◽  
Genetically Engineered ◽  
Clinical Isolates ◽  
Enzyme Expression ◽  
Data Set ◽  
Content Type ◽  
E Coli ◽  
Resistance Determinants ◽  
The Relationship

ABSTRACTWe recently investigated the pharmacokinetics-pharmacodynamics (PK-PD) of tazobactam in combination with ceftolozane against an isogenic CTX-M-15-producingEscherichia colitriplet set, genetically engineered to transcribe different levels ofblaCTX-M-15. The percentage of the dosing interval that tazobactam concentrations remained above a threshold (%Time>threshold) was identified as the PK-PD exposure measure that was most closely associated with efficacy. Moreover, the tazobactam concentration was dependent upon the enzyme transcription level. Given that the aforementioned strains were genetically engineered to transcribe a single β-lactamase enzyme and that clinical isolates typically produce multiple β-lactamase enzymes with various transcription levels, it is likely that the tazobactam threshold concentration is isolate/enzyme dependent. Our first objective was to characterize the relationship between the tazobactam %Time>threshold in combination with ceftolozane and efficacy using clinical isolates in anin vitroPK-PD infection model. Our second objective was to identify a translational relationship that would allow for the comodeling across clinical isolates. The initial challenge panel included four well-characterized β-lactamase-producingE. colistrains with variable enzyme expression and other resistance determinants. As evidenced byr2values of ranging from 0.90 to 0.99 for each clinical isolate, the observed data were well described by fitted functions describing the relationship between the tazobactam %Time>threshold and change in log10CFU from baseline; however, the data from the four isolates did not comodel well. The threshold concentration identified for each isolate ranged from 0.5 to 4 mg/liter. We identified an enabling translational relationship for the tazobactam threshold that allowed comodeling of all four clinical isolates, which was the product of the individual isolate's ceftolozane-tazobactam MIC value and 0.5. As evidenced by anr2value of 0.90, the transformed data were well described by a fitted function describing the relationship between tazobactam %Time>threshold and change in log10CFU from baseline. Due to these findings, the challenge panel was expanded to include three well-characterized β-lactamase-producingKlebsiella pneumoniaestrains with variable enzyme expression and other resistance determinants. The translational relationship for the tazobactam threshold that allowed for the comodeling of the fourE. coliisolates performed well for the expanded data set (seven isolates in total; fourE. coliand threeK. pneumoniae), as evidenced by anr2value of 0.84. This simple translational relationship is especially useful as it is directly linked toin vitrosusceptibility test results, which are used to guide the clinician's choice of drug and dosing regimen.

The in vitro O2′-methylation of RNA in the ribonucleoprotein precursor-particles from a Relaxed mutant of Escherichia coli

1968 ◽  
Vol 46 (1) ◽  
pp. 109-115 ◽  
Author(s):  
J. L. Nichols ◽  
B. G. Lane
Keyword(s):  
Escherichia Coli ◽  
Sodium Chloride ◽  
Snake Venom ◽  
Chloride Solution ◽  
Sodium Chloride Solution ◽  
E Coli ◽  
Adenosyl Methionine

Ribonucleoprotein "precursor-particles" were isolated from a methionine auxotroph of E. coli (RCrelaxed), which had been cultured under conditions of methionine deprivation. Following incubation of the "particles" in vitro with [methyl-14C]-S-adenosyl methionine, the ribonucleates were extracted from the particles, and precipitated from aqueous 2 M sodium chloride solution at 0°, before analysis for the incorporation of O2′-[methyl-14C]-substituents into the RNA. It was found that two of the four principal alkali-stable dinucleotides, known to be present in alkali hydrolysates of E. coli rRNA, contained O2′-[methyl-14C]-substituents: O2′Me-GpGp and O2′Me-CpCp, whereas the two remaining dinucleotides, N4,O2′diMe-CpCp and O2′Me-UpGp, did not contain significant radioactivity. It has been concluded that highly specific enzymic O2′-methylation of E. coli RNA can occur subsequent to polynucleotide synthesis. Allied studies are reported concerning the characterization of N4,O2′-[dimethyl-14C]-cytidine 5′-phosphate that was recovered from whole snake venom hydrolysates of E. coli rRNA, which had been labeled in vivo with [methyl-14C].

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

Антимикробная активность лиофилизированного водного извлечения Caragana jubata (Pall.) Poir.

2020 ◽  
Vol 54 (3) ◽  
pp. 42-44
Author(s):  
Павел Алексеевич Какорин ◽  
Татьяна Владимировна Фатеева ◽  
Ольга Ивановна Терешкина ◽  
Ирина Борисовна Перова ◽  
Галина Владиславовна Раменская ◽  
...  
Keyword(s):  
E Coli

На основании ранее проведенных исследований установлен профиль флавоноидов лиофилизированного водного извлечения, полученного из побегов C. jubata. В связи с тем, что, согласно данным литературы, флавоноиды являются потенциальными ингибиторами микроорганизмов, проведено изучение антимикробной активности лиофилизата в опытах in vitro с использованием скринигового метода определения антимикробной активности для препаратов растительного происхождения. При изучении бактериостатической и фунгистатической активности в опытах in vitro использовали метод двукратного серийного разведения препаратов в жидких питательных средах. В результате исследования лиофилизированного водного извлечения караганы гривастой установлено наличие умеренной антимикробной активности в отношении всех изученных штаммов патогенных микроорганизмов: грамположительных и грамотрицательных бактерий (S. aureus, E. coli, P. vulgaris, P. aeruginosa), дрожжеподобных и мицелиальных грибов (C. albicans, M. canis). Полученные данные позволяют рекомендовать лиофилизированное водное извлечение караганы гривастой для создания на его основе лекарственных форм наружного применения для лечения заболеваний кожи и слизистых оболочек, связанных с бактериальным воспалительным процессом.

Влияние in vitro изолированного и сочетанного с антибактериальными средствами применения бовгиалуронидазы азоксимер на целостность бактериальной биопленки и жизнеспосоность микроорганизмов

2020 ◽  
Vol 83 (2) ◽  
pp. 38-44
Author(s):  
Е. Ю. Тризна ◽  
Д. Р. Байдамшина ◽  
Александр А. Виницкий ◽  
А. Р. Каюмов
Keyword(s):  
E Coli

Исследована способность лиофилизата бовгиалуронидазы азоксимера («Лонгидаза») разрушать бактериальные биопленки S. aureus, E. faecalis, E. coli, а также сочетанное действие препарата с антибактериальными средствами. Показано, что 2 ч инкубации бовгиалуронидазы азоксимер в концентрации 750 – 1500 МЕ/мл вызывает двукратное снижение биомассы матрикса зрелых биопленок E. faecalis и E. coli, и на 60 % — S. aureus. Данный ферментный препарат не влияет на образование бактериальных биопленок. При сочетанном применении с антибактериальными средствами препарат повышает их эффективность в отношении бактерий в составе биопленок. Так, концентрация ципро-флоксацина и амоксициллина, необходимая для снижения количества КОЕ на 3 порядка в биопленке E. faecalis, в присутствии бовгиалуронидазы азоксимера снижается в 16 раз (p < 0,05). В присутствии фермента в 16 раз меньшие концентрации цефуроксима, фосфомицина, ципрофлоксацина и амикацина достаточны для снижения количества КОЕ на 3 порядка в биопленке E. coli (p < 0,05), и в значительно меньшей концентрации цефуроксим оказывает бактерицидное действие на клетки в биопленке S. aureus (p < 0,05). Вероятно, бовгиалуронидаза азоксимер увеличивает проникновение антибактериальных средств к клеткам бактерий в биопленке, что обеспечивает потенцирование их антибактериального эффекта. Такое действие ферментного препарата позволяет снизить дозу и повысить безопасность антибактериальных средств при сохранении их эффективности.

Effect of C6-Volatiles on Bioluminescent Plant Pathogens

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 557d-557
Author(s):  
Jennifer Warr ◽  
Fenny Dane ◽  
Bob Ebel
Keyword(s):  
Biological Activity ◽  
Bacterial Growth ◽  
Xanthomonas Campestris ◽  
Plant Pathogens ◽  
Genetically Engineered ◽  
The Other ◽  
Computer Assisted ◽  
Signal Molecules ◽  
Low Concentrations

C6 volatile compounds are known to be produced by the plant upon pathogen attack or other stress-related events. The biological activity of many of these substances is poorly understood, but some might produce signal molecules important in host–pathogen interactions. In this research we explored the possibility that lipid-derived C6 volatiles have a direct effect on bacterial plant pathogens. To this purpose we used a unique tool, a bacterium genetically engineered to bioluminesce. Light-producing genes from a fish-associated bacterium were introduced into Xanthomonas campestris pv. campestris, enabling nondestructive detection of bacteria in vitro and in the plant with special computer-assisted camera equipment. The effects of different C6 volatiles (trans-2 hexanal, trans-2 hexen-1-ol and cis-3 hexenol) on growth of bioluminescent Xanthomonas campestris were investigated. Different volatile concentrations were used. Treatment with trans-2 hexanal appeared bactericidal at low concentrations (1% and 10%), while treatments with the other volatiles were not inhibitive to bacterial growth. The implications of these results with respect to practical use of trans-2 hexanal in pathogen susceptible and resistant plants will be discussed.

Properties and Biotechnological Application of mutant Derivatives of Mini-intein PRP8 from Penicillium chrysogenum with Improved Control of C-terminal Processing

2019 ◽  
Vol 35 (6) ◽  
pp. 91-101
Author(s):  
F.A. Klebanov ◽  
S.E. Cheperegin ◽  
D.G. Kozlov
Keyword(s):  
Penicillium Chrysogenum ◽  
Protein Denaturation ◽  
Biologically Active ◽  
Cultivation Temperature ◽  
Target Proteins ◽  
E Coli ◽  
Complete Inactivation ◽  
Target Molecules ◽  
Proteins And Peptides

Mutant variants of mini-intein PRP8 from Penicillium chrysogenum (Int4b) with improved control of C-terminal processing were characterized. The presented variants can serve as a basis for self-removed polypeptide tags capable of carrying an affine label and allowing to optimize the process of obtaining target proteins and peptides in E. coli cells. They allow to synthesize target molecules in the composition of soluble and insoluble hybrid proteins (fusions), provide their afnne purification, autocatalytic processing and obtaining mature target products. The presented variants have a number of features in comparison with the known prototypes. In particular the mutant mini-intein Int4bPRO, containing the L93P mutation, has temperature-dependent properties. At cultivation temperature below 30 °C it allows the production of target molecules as part of soluble fusions, but after increasing of cultivation temperature to 37 °C it directs the most of synthesized fusions into insoluble intracellular aggregates. The transition of Int4bPRO into insoluble form is accompanied by complete inactivation of C-terminal processing. Further application of standard protein denaturation-renaturation procedures enable efficiently reactivate Int4bPRO and to carry out processing of its fusions in vitro. Two other variants, Int4b56 and Int4b36, containing a point mutation T62N or combination of mutations D144N and L146T respectively, have a reduced rate of C-terminal processing. Their use in E. coli cells allows to optimize the biosynthesis of biologically active target proteins and peptides in the composition of soluble fusions, suitable for afnne purification and subsequent intein-dependent processing without the use of protein denaturation-renaturation procedures. intein, fusion, processing, processing rate, gelonin The work was supported within the framework of the State Assignment no. 595-00003-19 PR.

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