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.

scholarly journals In Vitro Coliform Resistance to Bioactive Compounds in Urinary Infection, Assessed in a Lab Catheterization Model

2021 ◽  
Vol 11 (9) ◽  
pp. 4315
Author(s):  
Emanuel Vamanu ◽  
Laura Dorina Dinu ◽  
Cristina Mihaela Luntraru ◽  
Alexandru Suciu
Keyword(s):  
Urinary Tract ◽  
Bioactive Compounds ◽  
Urinary Tract Infections ◽  
Antimicrobial Effect ◽  
Biologically Active ◽  
Bacterial Strains ◽  
E Coli ◽  
Functional Product ◽  
Tract Infections

Bioactive compounds and phenolic compounds are viable alternatives to antibiotics in recurrent urinary tract infections. This study aimed to use a natural functional product, based on the bioactive compounds’ composition, to inhibit the uropathogenic strains of Escherichia coli. E. coli ATCC 25922 was used to characterize the IVCM (new in vitro catheterization model). As support for reducing bacterial proliferation, the cytotoxicity against a strain of Candida albicans was also determined (over 75% at 1 mg/mL). The results were correlated with the analysis of the distribution of biologically active compounds (trans-ferulic acid-268.44 ± 0.001 mg/100 g extract and an equal quantity of Trans-p-coumaric acid and rosmarinic acid). A pronounced inhibitory effect against the uropathogenic strain E. coli 317 (4 log copy no./mL after 72 h) was determined. The results showed a targeted response to the product for tested bacterial strains. The importance of research resulted from the easy and fast characterization of the functional product with antimicrobial effect against uropathogenic strains of E. coli. This study demonstrated that the proposed in vitro model was a valuable tool for assessing urinary tract infections with E. coli.

Comparison of immunomodulatory properties of purified lactoferrin, lactoperoxidase and β-casein in sheep

1998 ◽  
Vol 65 (4) ◽  
pp. 697-701 ◽  
Author(s):  
CHUN W. WONG ◽  
DENNIS L. WATSON ◽  
GEOFFREY O. REGESTER ◽  
GEOFFREY W. SMITHERS
Keyword(s):  
Biomedical Applications ◽  
Bovine Milk ◽  
Milk Proteins ◽  
Biologically Active ◽  
Immunological Effects ◽  
Bovine Milk Proteins ◽  
Proteins And Peptides

Bovine milk contains a variety of proteins and peptides that are biologically active (Ogra & Ogra, 1978; Duncan & McArthur, 1981; Newby et al. 1982; Juto, 1985; Stoeck et al. 1989; Mincheva-Nilsson et al. 1990; Watson, 1990; Barta et al. 1991; Politis et al. 1991; Fiat et al. 1993). Our laboratory has a long-term interest in some purified milk proteins, particularly lactoferrin (LF), lactoperoxidase (LP) and β-casein (β-CN), which have been shown to be immunologically significant. Some of our recent studies on these bovine milk proteins, particularly β-CN, indicated that their in vitro immunological effects did not always parallel their in vivo activities (Wong et al. 1996a, b; 1997a, b). This study was designed to investigate and compare the capacity of these purified bovine milk proteins to modulate a range of components that are vital to in vivo immune responses in sheep, with a view to providing further information on their potential in biomedical applications. To achieve this objective, a sensitive lymphatic cannulation model was employed that allows in vivo immune components and their functions to be measured in lymph collected under physiological conditions.

scholarly journals Recycling of Shiga Toxin 2 Genes in Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:NM

2007 ◽  
Vol 74 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Alexander Mellmann ◽  
Shan Lu ◽  
Helge Karch ◽  
Jian-guo Xu ◽  
Dag Harmsen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Integration Site ◽  
Hot Spot ◽  
Blot Hybridization ◽  
Enterohemorrhagic Escherichia Coli ◽  
Biologically Active ◽  
E Coli ◽  
Shiga Toxin 2

ABSTRACT Using colony blot hybridization with stx 2 and eae probes and agglutination in anti-O157 lipopolysaccharide serum, we isolated stx 2-positive and eae-positive sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) strains from initial stool specimens and stx-negative and eae-positive SF E. coli O157:NM strains from follow-up specimens (collected 3 to 8 days later) from three children. The stx-negative isolates from each patient shared with the corresponding stx 2-positive isolates fliC H7, non-stx virulence traits, and multilocus sequence types, which indicates that they arose from the stx 2-positive strains by loss of stx 2 during infection. Analysis of the integrity of the yecE gene, a possible stx phage integration site in EHEC O157, in the consecutive stx 2-positive and stx-negative isolates demonstrated that yecE was occupied in stx 2-positive but intact in stx-negative strains. It was possible to infect and lysogenize the stx-negative E. coli O157 strains in vitro using an stx 2-harboring bacteriophage from one of the SF EHEC O157:NM isolates. The acquisition of the stx 2-containing phage resulted in the occupation of yecE and production of biologically active Shiga toxin 2. We conclude that the yecE gene in SF E. coli O157:NM is a hot spot for excision and integration of Shiga toxin 2-encoding bacteriophages. SF EHEC O157:NM strains and their stx-negative derivatives thus represent a highly dynamic system that can convert in both directions by the loss and gain of stx 2-harboring phages. The ability to recycle stx 2, a critical virulence trait, makes SF E. coli O157:NM strains ephemeral EHEC that can exist as stx-negative variants during certain phases of their life cycle.

New Approaches for the Synthesis of Heterocyclic Compounds Corporating benzo[d]imidazole as Anticancer Agents, Tyrosine, Pim-1 Kinases Inhibitions and their PAINS Evaluations

2020 ◽  
Vol 20 ◽  
Author(s):  
Rafat M. Mohareb ◽  
Yara R. Milad ◽  
Bahaa M. Mostafa ◽  
Reem A. El-Ansary
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Anticancer Agents ◽  
Tyrosine Kinases ◽  
Heterocyclic Compounds ◽  
Cancer Cell Lines ◽  
Biologically Active ◽  
Strong Impact ◽  
Target Molecules

Background: Benzo[d]imidazoles are highly biologically active, in addition, they are considered as a class of heterocyclic compounds with many pharmaceutical applications. Objective: We are aiming in this work to synthesize target molecules not only possess anti-tumor activities but also kinase inhibitors. The target molecules were obtained starting from the benzo[d]imidazole derivatives followed by their heterocyclization reactions to produce anticancer target molecules. Methods: The 1-(1H-benzo[d]imidazol-2-yl)propan-2-one (3) and the ethyl 2-(1H-benzo[d]imidazol-2-yl)acetate (16) were used as the key starting material which reacted with salicylaldehyde to give the corresponding benzo[4,5]imidazo[1,2-a]quinoline derivatives. On the other hand, both of them were reacted with different reagents to give thiophene, pyran and benzo[4,5]imidazo[1,2-c]pyrimidine derivatives. The synthesized compounds were evaluated against the six cancer cell lines A549, HT-29, MKN-45, U87MG, and SMMC7721 and H460 together with inhibitions toward tyrosine kinases, c-Met kinase and prostate cancer cell line PC-3 were recorded using the standard MTT assay in vitro, with foretinib as the positive control. Results: Most of the synthesized compounds exhibited high inhibitions toward the tested cancer cell lines. In addition, tyrosine and Pim1 kinases inhibitions were performed for the most active compounds where variation of substituent through the aryl ring and heterocyclic ring afforded compounds with high activities. Our analysis showed that there is a strong correlation between structure of compound and substituents of target molecules. Conclusion: Our present research proved that the synthesized heterocyclic compounds with varieties of substituents has a strong impact through the activity of compounds. The evaluations through different cell lines and tyrosine kinases indicated that the compounds were excellent candidates as anticancer agents. This could encourage doing further research within this field for the building of compounds with high inhibitions.

scholarly journals Luteolin and abyssinone II as potential inhibitors of SARS-CoV-2: an in silico molecular modeling approach in battling the COVID-19 outbreak

2021 ◽  
Vol 45 (1) ◽  
Author(s):  
Mohammad Mahfuz Ali Khan Shawan ◽  
Sajal Kumar Halder ◽  
Md. Ashraful Hasan
Keyword(s):  
Molecular Dynamics ◽  
Root Mean Square ◽  
In Silico ◽  
Binding Free Energy ◽  
Biologically Active ◽  
Dynamics Simulation ◽  
Mean Square ◽  
Efficient Treatment ◽  
Target Proteins

Abstract Background At present, the entire world is in a war against COVID-19 pandemic which has gradually led us toward a more compromised “new normal” life. SARS-CoV-2, the pathogenic microorganism liable for the recent COVID-19 outbreak, is extremely contagious in nature resulting in an unusual number of infections and death globally. The lack of clinically proven therapeutic intervention for COVID-19 has dragged the world’s healthcare system into the biggest challenge. Therefore, development of an efficient treatment scheme is now in great demand. Screening of different biologically active plant-based natural compounds could be a useful strategy for combating this pandemic. In the present research, a collection of 43 flavonoids of 7 different classes with previously recorded antiviral activity was evaluated via computational and bioinformatics tools for their impeding capacity against SARS-CoV-2. In silico drug likeness, pharmacophore and Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) profile analysis of the finest ligands were carried out using DataWarrior, DruLiTo and admetSAR programs, respectively. Molecular docking was executed by AutoDock Vina, while molecular dynamics simulation of the target protein–ligand bound complexes was done using nanoscalable molecular dynamics and visual molecular dynamics software package. Finally, the molecular target analysis of the selected ligands within Homo sapiens was conducted with SwissTargetPredcition web server. Results Out of the forty-three flavonoids, luteolin and abyssinone II were found to develop successful docked complex within the binding sites of target proteins in terms of lowest binding free energy and inhibition constant. The root mean square deviation and root mean square fluctuation values of the docked complex displayed stable interaction and efficient binding between the ligands and target proteins. Both of the flavonoids were found to be safe for human use and possessed good drug likeness properties and target accuracy. Conclusions Conclusively, the current study proposes that luteolin and abyssinone II might act as potential therapeutic candidates for SARS-CoV-2 infection. In vivo and in vitro experiments, however, should be taken under consideration to determine the efficiency and to demonstrate the mechanism of action.

Expression of biologically active recombinant-derived chicken prolactin in Escherichia coli

1989 ◽  
Vol 3 (1) ◽  
pp. 15-21 ◽  
Author(s):  
M. C. Hanks ◽  
R. T. Talbot ◽  
H. M. Sang
Keyword(s):  
Escherichia Coli ◽  
Cdna Sequence ◽  
Expression Vector ◽  
Total Cell ◽  
Biologically Active ◽  
Cell Protein ◽  
Western Blots ◽  
E Coli ◽  
Ring Dove

ABSTRACT The putative chicken prolactin (chPRL) cDNA clone PRL101 was manipulated in vitro and cloned into the Escherichia coli expression vector pKK233-2 to produce a plasmid coding for recombinant-derived mature chPRL (R-chPRL). Expression of this manipulated cDNA sequence in E. coli resulted in the production of a 23 kDa protein which cross-reacted with specific chPRL antisera in Western blots. The partially purified protein stimulated ring dove crop sac mucosa to proliferate in a PRL bioassay, demonstrating that the R-chPRL was biologically active. R-chPRL was expressed at a level of approximately 1·5% of total cell protein.

scholarly journals Construction of a Novel Liver-Targeting Fusion Interferon by Incorporation of aPlasmodiumRegion I-Plus Peptide

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Xuemei Lu ◽  
Xiaobao Jin ◽  
Yanting Huang ◽  
Jie Wang ◽  
Juan Shen ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Current Treatment ◽  
Biologically Active ◽  
Liver Targeting ◽  
E Coli ◽  
Final Yield ◽  
Pcr Method ◽  
Overlapping Extension Pcr

Interferon alpha (IFNα) exerts a multiplicity of biological actions including antiviral, immunomodulatory, and antiproliferative effects. Administration of IFNαis the current treatment for chronic hepatitis B; however, therapy outcome has not been completely satisfactory. The systemic effects of IFNαmay account for its lowin vivobiological activity and multiple adverse events. The purpose of this study was to design a novel liver-targeting fusion interferon (IFN-CSP) by fusing IFNα2b with aPlasmodiumregion I-plus peptide, thus targeting the drug specifically to the liver. The DNA sequence encoding IFN-CSP was constructed using improved splicing by overlapping extension-PCR method, and then cloned into the pET-21b vector for protein expression inE. coliBL21 (DE3). The recombinant protein was expressed as a His-tagged protein and purified using a combination of Ni affinity and HiTrap affinity chromatography at a purity of over 95%. The final yield of biologically active IFN-CSP was up to 270 mg/L culture. The purified recombinant protein showed anti-HBV activity and liver-targeting potentialityin vitro. These data suggests that the novel fusion interferon IFN-CSP may be an excellent candidate as a liver-targeting anti-HBV agent.

scholarly journals A Truncated Soluble Bacillus Signal Peptidase Produced in Escherichia coli Is Subject to Self-Cleavage at Its Active Site

2000 ◽  
Vol 182 (20) ◽  
pp. 5765-5770 ◽  
Author(s):  
M. L. van Roosmalen ◽  
J. D. H. Jongbloed ◽  
A. Kuipers ◽  
G. Venema ◽  
S. Bron ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Peptidase ◽  
Soluble Form ◽  
Peptidase Activity ◽  
Mutant Proteins ◽  
E Coli ◽  
Amino Terminal ◽  
Complete Inactivation ◽  
High Level

ABSTRACT Soluble forms of Bacillus signal peptidases which lack their unique amino-terminal membrane anchor are prone to degradation, which precludes their high-level production in the cytoplasm ofEscherichia coli. Here, we show that the degradation of soluble forms of the Bacillus signal peptidase SipS is largely due to self-cleavage. First, catalytically inactive soluble forms of this signal peptidase were not prone to degradation; in fact, these mutant proteins were produced at very high levels in E. coli. Second, the purified active soluble form of SipS displayed self-cleavage in vitro. Third, as determined by N-terminal sequencing, at least one of the sites of self-cleavage (between Ser15 and Met16 of the truncated enzyme) strongly resembles a typical signal peptidase cleavage site. Self-cleavage at the latter position results in complete inactivation of the enzyme, as Ser15 forms a catalytic dyad with Lys55. Ironically, self-cleavage between Ser15 and Met16 cannot be prevented by mutagenesis of Gly13 and Ser15, which conform to the −1, −3 rule for signal peptidase recognition, because these residues are critical for signal peptidase activity.

scholarly journals Release of interleukin-12 in experimental Escherichia coli septic shock in baboons: relation to plasma levels of interleukin-10 and interferon- gamma

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 5144-5151 ◽  
Author(s):  
PM Jansen ◽  
TC van der Pouw Kraan ◽  
IW de Jong ◽  
G van Mierlo ◽  
J Wijdenes ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Septic Shock ◽  
Interferon Gamma ◽  
Biologically Active ◽  
Sublethal Dose ◽  
Interleukin 12 ◽  
Ifn Gamma ◽  
E Coli ◽  
Enhanced Production

Interleukin (IL)-12 is thought to be a key factor for the induction of interferon gamma (IFN-gamma), a cytokine essential for the lethal effects of endotoxin. We report here on the release of the nonfunctional subunit of IL-12, p40, as well as biologically active heterodimeric IL-12, p70, after administration of a lethal (n = 5) or sublethal (n = 8) dose of live Escherichia coli to baboons. Remarkably, on lethal challenge, peak levels of p40 were observed at 3 hours that were about twofold lower than those elicited after sublethal challenge (2,813 +/- 515 pg/mL v 4,972 +/- 732 pg/mL, P < .05). This disparity was also observed, although to a lesser extent, for IL-12 p70 antigen, of which maximum levels of 91 +/- 47 pg/mL and 151 +/- 41 pg/mL were measured 6 hours after a lethal or sublethal dose of E coli, respectively. Circulating p70 antigen correlated with IL-12 biologic activity (r = 0.869; P < .001). When comparing lethal to sublethal conditions, lower peak levels of IL-12 on lethal E coli sharply contrasted with higher levels of other proinflammatory cytokines, such as tumor necrosis factor (TNF)-alpha, IL-1beta, IL-6, and IL-8 observed in these animals. Lower IL-12 concentrations in the lethal group may have resulted in part from the enhanced production of IL-10, a known inhibitor of IL-12 synthesis in vitro, as peak levels of this cytokine 3 hours postchallenge inversely correlated with peak levels of IL-12, in particular p40 (r = -0.802; P < .01). Contrary to what might be expected if IFN-gamma were solely induced by IL-12, lethally challenged baboons generated threefold more IFN-gamma at 6 hours than those receiving a sublethal dose (P < .05). Moreover, higher levels of IFN- gamma were associated with lower p40/p70 ratios, suggesting that, in agreement with observations in vitro, IFN-gamma may have preferentially upregulated the release of p70 over p40. These data show that IL-12 is released in experimental septic shock in nonhuman primates and suggest that IL-10 and IFN-gamma are involved in the regulation of this release. Furthermore, this study indicates that the systemic release of IL-12 might be essential, but is not likely sufficient, to promote lethal production of IFN-gamma in sepsis.

Synthesis and antimicrobial activity of novel thienopyrimidine linked rhodanine derivatives

2019 ◽  
Vol 97 (2) ◽  
pp. 94-99 ◽  
Author(s):  
Nagaraju Kerru ◽  
Surya Narayana Maddila ◽  
Suresh Maddila ◽  
Sreedhar Sobhanapuram ◽  
Sreekantha B. Jonnalagadda
Keyword(s):  
Antimicrobial Activity ◽  
Condensation Reaction ◽  
Dilution Method ◽  
The Novel ◽  
E Coli ◽  
H Nmr ◽  
Target Molecules ◽  
Novel Target ◽  
C Nmr

This work presents the preparation of a new series of N-(substituted phenyl)-2-(4-oxo-5-(4-(thieno[2,3-d]-pyrimidin-4-yloxy)benzylidene)-2-thioxothiazolidin-3-yl)acetamide derivatives (8a–8l). A condensation reaction of thienopyrimidin-2-thioxothiazolidin-4-one derivative (5) with various 2-chloro-N-phenylacetamides (7a–7l) was employed to afford the new thienopyrimidine tagged rhodanine derivatives under acetone solvent in the presence of potassium carbonate (K2CO3). All of the novel target molecules were characterized by IR, 1H NMR, 13C NMR, and LC–MS spectral analyses and were screened for their in vitro antimicrobial activity by using the broth dilution method. Compounds 8c, 8g, and 8h found to have antibacterial potency against E. coli, B. subtilis, B. cereus, and K. pneumonia with minimum inhibitory concentrations (MICs) of 3.25–6.25 μg/mL compared with the standard Gentamicin. Compounds 8c and 8f demonstrated better antifungal potency (MIC = 3.25–6.25 μg/mL) against A. flavus, A. niger, P. marneffei, and C. albicans when compared with Fluconazole.

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