Localization and activity of recombinant streptavidin in cell fractions of Escherichia coli

2018 ◽  
Vol 18 (3) ◽  
pp. 69-76
Author(s):  
D V Zotova ◽  
N A Grudinina ◽  
O I Antimonova ◽  
M M Shavlovsky ◽  
D S Polyakov

The authors provide a genetic construct for obtaining recombinant biologically active wild type streptavidin with high yield. Addition of leader peptide and oligohistidine sequence to the streptavidin sequence makes it possible to isolate soluble streptavidin from the culture medium and from the soluble fraction. Temperature conditions for inducing of protein synthesis were found to have a significant effect on the distribution profile of streptavidin between fractions. The obtained protein product is not contaminated with endogenous biotin. The provided method excludes denaturation and renaturation steps which are necessary for isolation of the desired product from inclusion bodies but substantially reduce the yield. Thus, the provided approach allows to increase the yield of biologically highly active strepatividin which is capable of binding biotin and biotin-containing compounds and can be used for various practical purposes.

2018 ◽  
Vol 28 (1) ◽  
pp. 28-36
Author(s):  
Sureyya Mert Selimoglu ◽  
Murat Kasap ◽  
Gurler Akpinar ◽  
Aynur Karadenizli ◽  
Abdul Mounem Wis ◽  
...  

Human creatine kinase MB (hCKMB) is one of the most preferred biomarkers used for the diagnosis of acute coronary syndrome due to its high sensitivity and specificity. The increasing need for highly purified and biologically active hCKMB in the field of diagnostics makes its production valuable. Currently, the production of hCKMB is mainly achieved in methylotrophic yeast, <i>Pichia pastoris</i>, because the production in <i>Escherichia coli</i> is challenging and generally yields an inactive enzyme with a low quantity. With the aim of finding the best way for the high-yield production of active hCKMB in <i>E. coli</i>, an efficient strategy was developed using a construct allowing tandem expression of each subunit with 2 different tags. The strategy allowed the efficient expression and separate characterization of each subunit and 1-step purification of the heterodimeric protein into homogeneity. The heterodimeric protein displayed more than 11-fold greater specific activity than the commercially available one. The production strategy described in this study shows a clear advantage over the currently used ones and can be made available not only for laboratory scale production but also for commercial production. Our study is also a well-suited example for the studies in which novel protein expression strategies are needed to achieve greater yields with higher purities.


1960 ◽  
Vol XXXV (II) ◽  
pp. 225-234 ◽  
Author(s):  
R. Bourrillon ◽  
R. Got ◽  
R. Marcy

ABSTRACT A new method for preparation of Human Menopausal Gonadotrophin involves successively alcoholic precipitation, kaolin adsorption and chromatography on ion exchangers. A highly active material is obtained which corresponds to 1 mg per litre of urine and has an activity of 1 mouse uterus unit at a dose of 0.003 mg. This gonadotrophin possesses both follicle stimulating and luteinizing activities in hypophysectomized female rats, by histological study. It contains 13 % hexose, 10% hexosamine and 8.5 % sialic acid. A further purification, by zone electrophoresis on starch, gives a final product, biologically active at 0.001 mg, which behaves as an homogenous substance in free electrophoresis with mobility −4.76 × 10−5 at pH 8.6.


2018 ◽  
Author(s):  
Sreerangappa Ramesh ◽  
Kiran Indukuri ◽  
Olivier Riant ◽  
Damien Debecker

<p>Sodium aluminate is presented as a highly active heterogeneous catalyst able to convert a range of alcohols into the corresponding mixed carbonate esters, in high yield and under green conditions. The reaction is carried out using dimethyl carbonate both as a reactant and solvent, at 90°C. Allylic, aliphatic and aromatic alcohols are converted in good yields. The solid catalyst is shown to be truly heterogeneous, resistant to leaching, and recyclable. </p>


2020 ◽  
Vol 17 (2) ◽  
pp. 82-90 ◽  
Author(s):  
Ghodsi Mohammadi Ziarani ◽  
Fatemeh Mohajer ◽  
Zohreh kheilkordi

Background: Natural products have been received attention due to their importance in human life as those are biologically active. In this review, there are some reports through different methods related to the synthesis of the indolizidine 195B which was extracted from poisonous frog; however, due to respect nature, the synthesis of natural compounds such as indolizidine has been attracted much attention among scientists and researchers. Objective: This review discloses the procedures and methods to provide indolizidine 195B from 1989 to 2018 due to their importance as a natural product. Conclusion: There are several methods to give rise to the indolizidine 195B as a natural product that is highly active from the biological perspective in pharmaceutical chemistry. In summary, many protocols for the preparations of indolizidine 195B from various substrates, several reagents, and conditions have been reported from different aromatic and aliphatic.


2019 ◽  
Vol 16 (7) ◽  
pp. 953-967 ◽  
Author(s):  
Ghodsi M. Ziarani ◽  
Fatemeh Mohajer ◽  
Razieh Moradi ◽  
Parisa Mofatehnia

Background: As a matter of fact, nitrogen as a hetero atom among other atoms has had an important role in active biological compounds. Since heterocyclic molecules with nitrogen are highly demanded due to biological properties, 4-phenylurazole as a compound containing nitrogen might be important in the multicomponent reaction used in agrochemicals, and pharmaceuticals. Considering the case of fused derivatives “pyrazolourazoles” which are highly applicable because of their application for analgesic, antibacterial, anti-inflammatory and antidiabetic activities as HSP-72 induction inhibitors (I and III) and novel microtubule assembly inhibitors. It should be mentioned that spiro-pyrazole also has biological activities like cytotoxic, antimicrobial, anticonvulsant, antifungal, anticancer, anti-inflammatory, and cardiotonic activities. Objective: Urazole has been used in many heterocyclic compounds which are valuable in organic syntheses. This review disclosed the advances in the use of urazole as the starting material in the synthesis of various biologically active molecules from 2006 to 2019. Conclusion: Compounds of urazole (1,2,4-triazolidine-3,5-dione) are the most important molecules which are highly active from the biological perspective in the pharmaceuticals as well as polymers. In summary, many protocols for preparations of the urazole derivatives from various substrates in multi-component reactions have been reported from different aromatic and aliphatic groups which have had carbonyl groups in their structures. It is noted that several catalysts have been synthesized to afford applicable molecules with urazole scaffolds. In some papers, being environmentally friendly, short time reactions and high yields are highlighted in the protocols. There is a room to synthesize new catalysts and perform new reactions by manipulating urazole to produce biologically active compounds, even producing chiral urazole component as many groups of chiral urazole compounds are important from biological perspective.


Antioxidants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 918
Author(s):  
Nóra Emilia Nagybákay ◽  
Michail Syrpas ◽  
Vaiva Vilimaitė ◽  
Laura Tamkutė ◽  
Audrius Pukalskas ◽  
...  

The article presents the optimization of supercritical CO2 extraction (SFE-CO2) parameters using response surface methodology (RSM) with central composite design (CCD) in order to produce single variety hop (cv. Ella) extracts with high yield and strong in vitro antioxidant properties. Optimized SFE-CO2 (37 MPa, 43 °C, 80 min) yielded 26.3 g/100 g pellets of lipophilic fraction. This extract was rich in biologically active α- and β-bitter acids (522.8 and 345.0 mg/g extract, respectively), and exerted 1481 mg TE/g extract in vitro oxygen radical absorbance capacity (ORAC). Up to ~3-fold higher extraction yield, antioxidant recovery (389.8 mg TE/g pellets) and exhaustive bitter acid extraction (228.4 mg/g pellets) were achieved under the significantly shorter time compared to the commercially used one-stage SFE-CO2 at 10–15 MPa and 40 °C. Total carotenoid and chlorophyll content was negligible, amounting to <0.04% of the total extract mass. Fruity, herbal, spicy and woody odor of extracts could be attributed to the major identified volatiles, namely β-pinene, β-myrcene, β-humulene, α-humulene, α-selinene and methyl-4-decenoate. Rich in valuable bioactive constituents and flavor compounds, cv. Ella hop SFE-CO2 extracts could find multipurpose applications in food, pharmaceutical, nutraceutical and cosmetics industries.


2021 ◽  
Vol 12 ◽  
pp. 204173142110086
Author(s):  
Jun Yong Kim ◽  
Won-Kyu Rhim ◽  
Yong-In Yoo ◽  
Da-Seul Kim ◽  
Kyoung-Won Ko ◽  
...  

Exosomes derived from mesenchymal stem cells (MSCs) have been studied as vital components of regenerative medicine. Typically, various isolation methods of exosomes from cell culture medium have been developed to increase the isolation yield of exosomes. Moreover, the exosome-depletion process of serum has been considered to result in clinically active and highly purified exosomes from the cell culture medium. Our aim was to compare isolation methods, ultracentrifuge (UC)-based conventional method, and tangential flow filtration (TFF) system-based method for separation with high yield, and the bioactivity of the exosome according to the purity of MSC-derived exosome was determined by the ratio of Fetal bovine serum (FBS)-derived exosome to MSC-derived exosome depending on exosome depletion processes of FBS. The TFF-based isolation yield of exosome derived from human umbilical cord MSC (UCMSC) increased two orders (92.5 times) compared to UC-based isolation method. Moreover, by optimizing the process of depleting FBS-derived exosome, the purity of UCMSC-derived exosome, evaluated using the expression level of MSC exosome surface marker (CD73), was about 15.6 times enhanced and the concentration of low-density lipoprotein-cholesterol (LDL-c), known as impurities resulting from FBS, proved to be negligibly detected. The wound healing and angiogenic effects of highly purified UCMSC-derived exosomes were improved about 23.1% and 71.4%, respectively, with human coronary artery endothelial cells (HCAEC). It suggests that the defined MSC exosome with high yield and purity could increase regenerative activity.


2020 ◽  
Vol 12 (2) ◽  
pp. 166-173
Author(s):  
Anastasiia Obradovych ◽  
Liliia Vasina ◽  
Nadiia Zholobak

Cerium dioxide nanoparticles (CeO2 NP) have significant catalytic and antioxidant properties due to their ability to inhibit the development of free-radical reactions due to a combination of superoxidedismutase and catalase mimetic activities. This nanopreparation has a significant antibacterial effect, but is low-toxic to unicellular yeast Saccharomyces cerevisiae. Therefore, it is relevant and appropriate to study the effect of this compound on other groups of yeast, in particular members of the genus Rhodotorula spp., which are characterized by high growth rate, unpretentiousness to the component composition of the culture medium, the ability to form a variety of valuable biologically active compounds, as well as the ability to synthesize proteins, lipids, carotenoids and exopolysaccharides in the complex. The report that microorganisms of this genus have been identified as part of the normal fish microbiota (Abramis brama, Rutilus rutilus, Perca fluviatilis, Oreochromis niloticus) expands the range of their use, either as probiotics in general or their individual cellular and extracellular components as prebiotics. In this work, the effect of different concentrations of CeO2 NP nanopreparation on the production parameters of Rhodotorula glutinis was studied, as well as their antagonistic properties after saturation of nanocerium were tested. To detect the optimal concentration of nanopreparation of cerium dioxide, R. glutinis was cultured for 120 h at 28 0C and aerated at 160 rpm on Saburo medium with the addition of citrate-stabilized CeO2 NP with a particle size of 1-2 nm in the following concentrations: 100 mM, 10 mM, 1 mM, 0,1 mM, 0,01 mM. The control was R. glutinis grown on Saburo medium, experimental controls were represented by microorganisms in the culture medium of which citrate and full-size cerium (CeCl3) were added. It was found that most of the studied concentrations of CeO2 NP (except for the highest) are not toxic to these carotenogenic yeasts. The presence of 10 mM CeO2 NP stimulates the growth of the culture, while the increase in biomass is increased by fourfold compared to the control. There was also an increase in the content of basic metabolites – protein by 1,2 times, lipids by 2,5 times and valuable biologically active compounds - carotenoids by 20% and exopolysaccharides by 2,2 times. However, under these conditions, no significant changes in catalase and superoxidase activities were registered. Examination of the antagonistic properties of the CeO2 NP-enriched culture of R. glutinis by delayed antagonism by the method of perpendicular strokes against gram-positive B. subtilis and gram-negative P. syringae bacteria showed the presence of lysis zones up to 17 and 25 mm, respectively, in contrast to their complete or complete absence (2 mm), noted in the study of control samples. A study of the resistance of CeO2 NP-enriched culture of R. glutinis to natural inhibitors of the digestive tract showed resistance of microorganisms to gastric juice (the amount of CFU is 1,4 times higher than control values) and bile (recorded a 5-fold increase in survival of microorganisms). The obtained results testify to the prospects of using nanocerium for mobilization of growth, enzymatic, antagonistic activities of R. glutinis culture.


2020 ◽  
Vol 9 (1) ◽  
pp. 919-923

Biginelli, an important multicomponent reaction provides an avenue for the synthesis of different biologically active heterocyclic compounds. During the past decade, one pot multicomponent reactions have attracted the attention of organic and medicinal chemists due to high atom economy, time and enery saving convergent nature. The present manuscript reports a simple one pot three component synthesis of 3, 4-dihydroprimidin-2(1H)-thiones from various diversely substituted aldehydes, ethyl acetoacetate and thiourea using a orange peel powder as a natural catalyst on ultrasonic irradiation in aqueous medium as the solvent. The advantages of this reaction are less reaction time, high yield, easy availability of the catalyst and green nature.


2020 ◽  
Author(s):  
Nicholas Jose ◽  
mikhail Kovalev ◽  
Eric Bradford ◽  
Artur Schweidtmann ◽  
Hua Chun Zeng ◽  
...  

Novel materials are the backbone of major technological advances. However, the development and wide-scale introduction of new materials, such as nanomaterials, is limited by three main factors—the expense of experiments, inefficiency of synthesis methods and complexity of scale-up. Reaching the kilogram scale is a hurdle that takes years of effort for many nanomaterials. We introduce an improved methodology for materials development, combining state-of-the-art techniques—multi-objective machine learning optimization, high yield microreactors and high throughput analysis. We demonstrate this approach by efficiently developing a kg per day reaction process for highly active antibacterial ZnO nanoparticles. The proposed method has the potential to significantly reduce experimental costs, increase process efficiency and enhance material performance, which culminate to form a new pathway for materials discovery.


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