Structural analysis and in vitro processing to p5 rRNA of a 9S RNA molecule isolated from an rne mutant of E. coli

While DNA-dependent RNA polymerase represents one of the key enzymes involved in transcription and ultimately in gene expression in procaryotic and eucaryotic cells, little progress has been made towards elucidation of its 3-D structure at the molecular level over the past few years. This is mainly because to date no 3-D crystals suitable for X-ray diffraction analysis have been obtained with this rather large (MW ~500 kd) multi-subunit (α2ββ'ζ). As an alternative, we have been trying to form ordered arrays of RNA polymerase from E. coli suitable for structural analysis in the electron microscope combined with image processing. Here we report about helical polymers induced from holoenzyme (α2ββ'ζ) at low ionic strength with 5-7 mM MnCl2 (see Fig. 1a). The presence of the ζ-subunit (MW 86 kd) is required to form these polymers, since the core enzyme (α2ββ') does fail to assemble into such structures under these conditions.

Download Full-text

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

10.26434/chemrxiv.7725002.v1 ◽

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.

Download Full-text

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

Химико-фармацевтический журнал ◽

10.30906/0023-1134-2020-54-3-42-44 ◽

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.

Download Full-text

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

Экспериментальная и клиническая фармакология ◽

10.30906/0869-2092-2020-83-2-38-44 ◽

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.

Download Full-text

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

Biotekhnologiya ◽

10.21519/0234-2758-2019-35-6-91-101 ◽

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.

Download Full-text

In Vitro Holdase Activity of E. coli Small Heat-Shock Proteins IbpA, IbpB and IbpAB: A Biophysical Study with Some Unconventional Techniques

Protein and Peptide Letters ◽

10.2174/0929866521666131224094408 ◽

2014 ◽

Vol 21 (6) ◽

pp. 564-571 ◽

Cited By ~ 4

Author(s):

Sourav Roy ◽

Monobesh Patra ◽

Suman Nandy ◽

Milon Banik ◽

Rakhi Dasgupta ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Proteins ◽

Small Heat Shock Proteins ◽

E Coli ◽

Biophysical Study ◽

Shock Proteins

Download Full-text

Substituted 4-Formyl-2H-chromen-2-ones: Their Reaction with N-(2,3,4,6-Tetra-Oacetyl- β-D-galactopyranosyl)thiosemicarbazide, Antibacterial and Antifungal Activity of Their Thiosemicarbazone Products

Current Organic Chemistry ◽

10.2174/1385272824999200812132256 ◽

2020 ◽

Vol 24 (19) ◽

pp. 2272-2282

Author(s):

Vu Ngoc Toan ◽

Nguyen Minh Tri ◽

Nguyen Dinh Thanh

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Candida Albicans ◽

Selenium Dioxide ◽

Antibacterial And Antifungal Activity ◽

E Coli ◽

Antibacterial And Antifungal Activities ◽

Alkyl Ethers ◽

Antibacterial And Antifungal

Several 6- and 7-alkoxy-2-oxo-2H-chromene-4-carbaldehydes were prepared from corresponding alkyl ethers of 6- and 7-hydroxy-4-methyl-2-oxo-2H-chromen-2-ones by oxidation using selenium dioxide. 6- and 7-Alkoxy-4-methyl-2H-chromenes were obtained with yields of 57-85%. Corresponding 4-carbaldehyde derivatives were prepared with yields of 41-67%. Thiosemicarbazones of these aldehydes with D-galactose moiety were synthesized by reaction of these aldehydes with N-(2,3,4,6-tetra-O-acetyl-β-Dgalactopyranosyl) thiosemicarbazide with yields of 62-74%. These thiosemicarbazones were screened for their antibacterial and antifungal activities in vitro against bacteria, such as Staphylococcus aureus, Escherichia coli, and fungi, such as Aspergillus niger, Candida albicans. Several compounds exhibited strong inhibitory activity with MIC values of 0.78- 1.56 μM, including 8a (against S. aureus, E. coli, and C. albicans), 8d (against E. coli and A. niger), 9a (against S. aureus), and 9c (against S. aureus and C. albicans).

Download Full-text

Acetate Kinase (AcK) is Essential for Microbial Growth and Betel-derived Compounds Potentially Target AcK, PhoP and MDR Proteins in M. tuberculosis, V. cholerae and Pathogenic E. coli: An in silico and in vitro Study

Current Topics in Medicinal Chemistry ◽

10.2174/1568026619666190121105851 ◽

2019 ◽

Vol 18 (31) ◽

pp. 2731-2740 ◽

Cited By ~ 3

Author(s):

Sandeep Tiwari ◽

Debmalya Barh ◽

M. Imchen ◽

Eswar Rao ◽

Ranjith K. Kumavath ◽

...

Keyword(s):

Broad Spectrum ◽

In Silico ◽

Pathogenic Bacteria ◽

In Vitro Study ◽

Docking Studies ◽

Acetate Kinase ◽

E Coli ◽

Pathogenic Escherichia Coli ◽

Novel Target

Background: Mycobacterium tuberculosis, Vibrio cholerae, and pathogenic Escherichia coli are global concerns for public health. The emergence of multi-drug resistant (MDR) strains of these pathogens is creating additional challenges in controlling infections caused by these deadly bacteria. Recently, we reported that Acetate kinase (AcK) could be a broad-spectrum novel target in several bacteria including these pathogens. Methods: Here, using in silico and in vitro approaches we show that (i) AcK is an essential protein in pathogenic bacteria; (ii) natural compounds Chlorogenic acid and Pinoresinol from Piper betel and Piperidine derivative compound 6-oxopiperidine-3-carboxylic acid inhibit the growth of pathogenic E. coli and M. tuberculosis by targeting AcK with equal or higher efficacy than the currently used antibiotics; (iii) molecular modeling and docking studies show interactions between inhibitors and AcK that correlate with the experimental results; (iv) these compounds are highly effective even on MDR strains of these pathogens; (v) further, the compounds may also target bacterial two-component system proteins that help bacteria in expressing the genes related to drug resistance and virulence; and (vi) finally, all the tested compounds are predicted to have drug-like properties. Results and Conclusion: Suggesting that, these Piper betel derived compounds may be further tested for developing a novel class of broad-spectrum drugs against various common and MDR pathogens.

Download Full-text