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Cleavage Of Structural Proteins During Assembly Of Head Of Bacteriophage-T4
Abstract
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
... The cells were serum-starved for 4 h and then stimulated with 1 µM LPA or 1 µM PMA (these concentrations were determined in preliminary experiments) for the times indicated; after this incubation, cells were washed twice with ice-cold phosphate-buffered saline and lysed [66] for 1 h on an ice bath; the lysates were centrifuged at 12,700× g for 15 min, and proteins contained in supernatants were denatured with Laemmli sample buffer [69] and separated by SDS-polyacrylamide gel electrophoresis. Proteins were electrotransferred onto polyvinylidene difluoride membranes, and immunoblotting was performed. ...
... The extracts were centrifuged, and the supernatants were incubated overnight with protein A agarose and the anti-GFP antise-rum generated in our laboratory. Samples were washed five times, and the pellets were denaturalized with sample buffer [69]. Proteins were separated using SDS-polyacrylamide gel electrophoresis, electrotransferred onto nitrocellulose membranes, and exposed for 24 h. ...
... Cell lysates were centrifuged for 15 min at 4 °C, and the supernatant was added to a slurry of anti-GFP agarose beads and incubated with constant movement for 4 h at 4 °C. The pellets were washed five times with lysis buffer and solubilized with Laemmli sample buffer [69], and the samples were resolved using SDS-polyacrylamide gel electrophoresis. Finally, the bands corresponding to the LPA3-GFP construct (≈70 kDa, identified by Western blotting) were excised and sent to the Taplin Mass Spectrometry Facility (Harvard Medical School, Cambridge, MA, USA), where the analysis was performed. ...
LPA3 receptors were expressed in TREx HEK 293 cells, and their signaling and phosphorylation were studied. The agonist, lysophosphatidic acid (LPA), increased intracellular calcium and ERK phosphorylation through pertussis toxin-insensitive processes. Phorbol myristate acetate, but not LPA, desensitizes LPA3-mediated calcium signaling, the agonists, and the phorbol ester-induced LPA3 internalization. Pitstop 2 (clathrin heavy chain inhibitor) markedly reduced LPA-induced receptor internalization; in contrast, phorbol ester-induced internalization was only delayed. LPA induced rapid β-arrestin–LPA3 receptor association. The agonist and the phorbol ester-induced marked LPA3 receptor phosphorylation, and phosphorylation sites were detected using mass spectrometry. Phosphorylated residues were detected in the intracellular loop 3 (S221, T224, S225, and S229) and in the carboxyl terminus (S321, S325, S331, T333, S335, Y337, and S343). Interestingly, phosphorylation sites are within sequences predicted to constitute β-arrestin binding sites. These data provide insight into LPA3 receptor signaling and regulation.
... After separating all the protein subfractions (BMC mix) and pre-stained molecular weight marker (10-250 kDa) using Laemmli's SDS-PAGE [23], under the following conditions: 15% (w.v −1 ) polyacrylamide small gel, 65 V/30 min-90 V/120 min] in a vertical Bio-Rad Mini-Protean ® III cell, they were electro-transferred (conditions: semidry blotting, 1.2 mA cm 2 , 80 mA, 1.5 h) to PVDF membranes (0.45 µm) using a blotting system (Thermo Scientific TM Owl TM HEP-1 series). The blotted membranes were firstly activated (absolute methanol, 30 seg) then blocked with TBST [0.05 M Tris, 0.15 M NaCl, 2% Tween-20 (pH 7.42)] for two minutes}, then with less-detergent (Tween-20, 0.05% v.v −1 ) TBST, and further cut into smaller vertical strips (1 line per strip). ...
... The non-IgE mediated immunogenic potential of BMC fractions other than β-CN in ASD patients, has rarely been reported. Here, SDS-PAGE [23] and immunoblotting [24] of the BMC mix [α-CN (Sigma C6780) + β-CN (Sigma C6905), >90% pure] revealed quite interesting yet unexpected results: ...
Severe gastrointestinal symptoms (GIS) and food hypersensitivity are tightly associated in young individuals with autism spectrum disorders (ASD). Here, we explored the relationship of GIS (gastrointestinal severity index, ROMA IV criteria, Bristol scale), ASD-like behaviors (Childhood Autism Rating Scale), and certain sociodemographic/clinical traits (epidemiological survey) with serum immunoreactivity (IgG, IgA, IgE titers) towards bovine milk caseins (BMC; by ELISA) and subfractions (by immunoblotting) in thirty-one pediatric patients (~3-15 y, 77% male) with mild-to-severe GIS and ASD-like behaviors. In total, 42%, 25%, and 23% of all participants exhibited no (IgG−/IgA−), mono (IgG+/IgA−), or dual (IgG+/IgA+) immunoreactivity to BMC, respectively; the trend was significantly associated with the severity of the GIS and ASD-like behaviors, regurgitations, and self-reported allergies (OR: 1 → (1.9-3.1) → 13.5-16.0)]. No IgE+ response to BMC was found. Dual responders were α > κ > β-casein, though nonspecific reactivity to other protein fractions was also observed. The IgA+ > IgG+ but not IgE+ response to BMC (mainly α-casein) seems to be related to the severity of GIS and ASD-like behaviors, although a larger number of ASD patients are needed to draw a causal association.
... The protein composition was determined by using SDSpolyacrylamide gel electrophoresis by Laemmli (1970). The results obtained by electrophoretic analyses were then quantified by gel scanning and densitometric analysis using BioVision (W ilber, Germany) software. ...
Background: Pea (Pisum sativum L.) provides one of the best solutions for the lack of plant-based protein. The different agroecological conditions and the difference in seed color and type can affect seed protein content, composition and agronomic traits. Methods: A two-year trial on two European sites was done using an augmented block design. Agronomic traits and seed protein content were determined for each plot. Pearson’s correlation coefficients and multivariate analysis were done to analyze the structure and pea traits. Electrophoresis was done to investigate variations in protein composition. Result: The results of multivariate analysis showed the separation of pigmented seeds from non-pigmented seeds, with no clear grouping concerning seed type. The protein composition differed between seeds of different colors. Environmental factors had a significant impact on the duration of flowering, the number of pods and seeds and seed weight per plant.
... To characterize the efficiency of separation, freeze-dried isolated protein fractions were analyzed by SDS-PAGE in denaturing conditions according to Laemmli (1970), with slight modifications. Experiments were performed in non-reducing and reducing conditions using βmercaptoethanol. ...
Faba bean ingredients are rich in proteins and good sources of calcium (Ca), although containing phytic acid (PA) molecules. PA, a polyphosphate compound, can affect the bioavailability of minerals/proteins through complex formation. This study evaluates the impact of two extraction processes, Alkaline Extraction-IsoElectric Precipitation (AE-IEP) and Sequential Extraction (SE), on the ability of faba bean globulin systems to bind added calcium ions. Increasing concentrations of CaCl 2 were introduced into 2.5% (w/v) protein dispersions at pHs 4.5, 5.5, 6.5, and 7.5, and free Ca monitored. Near the isoelectric point of globulin (pH~4-5), Ca binding capacity was found to be low. At higher pHs, significant Ca chelation occurred, initially attributed to free PA binding sites, resulting in the formation of insoluble complexes and subsequent protein precipitation. The AE-IEP globulin fraction exhibited a higher Ca binding capacity than the SE globulin, attributed to its higher PA and lower initial Ca concentrations.
... The homogeneity of the purified protein and its molecular weight were determined by SDS-PAGE technique [18]. Purified protein was loaded onto 12% SDS-PAGE gel along with molecular weight markers and a graph for log molecular weight (log Mw) v. relative mobility (Rf) was plotted. ...
Superoxide dismutase is an important enzyme with various therapeutic applications. Search of a new source of superoxide dismutase with novel properties has significant importance. The current work reports purification of a novel superoxide dismutase enzyme with unique characteristics. A copper zinc superoxide dismutase (Cu-Zn SOD) was purified and characterized from Cicer arietinum L. seedlings germinated under aluminium (Al +3) stress. The specific activity of purified protein was 158 units/mg with 28 fold purification. The superoxide dismutase is a homodimeric protein with ap-prox subunit molecular weight of 33.27 kDa. The enzyme is identified as Cu-Zn category of superoxide dismutase, reflected by H 2 O 2 induced inhibition of in-gel activity and presence of quantifiable copper and zinc ions. The optimum pH range for purified Cu-Zn SOD activity was observed within 6.5-8.5 (highest at pH 8.0) and the pH stability was in the range of 6.0-8.5. The enzyme was more stable at low temperature (below 30°C) and the K m of purified Cu-Zn SOD for ri-boflavin as substrate was 10.16 ± 2.5 M. The N-terminal amino acid sequence showed homology at conserved residues with other plant Cu-Zn SODs.
... The resulting aliquots of the enzyme's solution were stored at −80 • C for further experiments. We have measured the protein concentration according to the Bradford assay and determined the enzyme purity through SDS PAGE [24,25]. ...
1,2,4-Triazole derivatives have a wide range of biological activities. The most well-known drug that contains 1,2,4-triazole as part of its structure is the nucleoside analogue ribavirin, an antiviral drug. Finding new nucleosides based on 1,2,4-triazole is a topical task. The aim of this study was to synthesize ribosides and deoxyribosides of 1,2,4-triazole-3-thione derivatives and test their antiviral activity against herpes simplex viruses. Three compounds from a series of synthesized mono- and disubstituted 1,2,4-triazole-3-thione derivatives were found to be substrates for E. coli purine nucleoside phosphorylase. Of six prepared nucleosides, the riboside and deoxyriboside of 3-phenacylthio-1,2,4-triazole were obtained at good yields. The yields of the disubstituted 1,2,4-triazol-3-thiones were low due to the effect of bulky substituents at the C3 and C5 positions on the selectivity of enzymatic glycosylation for one particular nitrogen atom in the triazole ring. The Academic Editor: Jesús Fernandez Lucas Received: 10 May 2024 Revised: 18 June 2024 Accepted: 19 June 2024 Published: 24 June 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). results of cytotoxic and antiviral studies on acyclovir-sensitive wild-type strain HSV-1/L2(TK+) and acyclovir-resistant strain (HSV-1/L2/RACV) in Vero E6 cell culture showed that the incorporation of a thiobutyl substituent into the C5 position of 3-phenyl-1,2,4-triazole results in a significant increase in the cytotoxicity of the base and antiviral activity. The highest antiviral activity was observed in the 3-phenacylthio-1-(β-D-ribofuranosyl)-1,2,4-triazole and 5-butylthio-1-(2-deoxy-β-D-ribofuranosyl)3-phenyl-1,2,4-triazole nucleosides, with their selectivity indexes being significantly higher than that of ribavirin. It was also found that with the increasing lipophilicity of the nucleosides, the activity and toxicity of the tested compounds increased.
... Total soluble proteins were separated from the pellet by centrifugation at 13000 rpm at 4 °C for 10 minutes. The pellet (insoluble fraction) was resuspended in an equivalent volume of Tris-HCl 20 mM, pH 8.0 Proteins from soluble and insoluble fractions were analyzed by SDS-PAGE using a 12.6% gel, followed by Coomassie blue staining (Laemmli, 1970). ...
SUMO (Small Ubiquitin-related Modifier) protease is widely used for recombinant protein expression, but obtaining the protein from derivative sources is challenging due to the degradation during extraction and high costs. In a previous report, we expressed the human SUMO protease’s active region (SENP2) fused with thioredoxin (Trx-SENP2) in E. coli BL21 (DE3). The recombinant fusion protein was soluble. In this study, we present the results of purification and activity evaluation of soluble Trx-SENP2. The enzyme was effectively purified through a single step Ni2 affinity chromatography. Trx-SENP2 was bound to the column in a binding buffer containing 50 mM PBS, 500 mM NaCl, 50 mM imidazol, pH 7.4 and eluted in the basic buffer containing 300 mM imidazol. The purity of the enzyme obtained after purification reached over 98% and yield of Trx-SENP2 was 361 mg/liter of bacterial culture. Importantly, the Trx-SENP2 was biologically active and capable of degrading recombinant SUMO-fused proteins. The protease’s bioactivity, as measured by its ability to cleave SUMO-IL11 into SUMO and IL11 fragments, was 3.33 × 105 U/mg comparable to commercial enzymes. Moreover, the Trx-SENP2 was found to be effective activity under a variety of conditions, including different buffers with high salt and imidazole concentrations and a wide range of pH values. Thus, the properties are useful and easy for the application of the enzyme to cleave recombinant SUMO-fused protein in the process of purification to obtain target proteins. The present study introduces a highly active and easily applicable enzyme for research in recombinant DNA technology.
... SDS-PAGE 10% was applied to analyze the purity level and estimate the isolated protein's molecular weight. SDS-PAGE was prepared based on Laemli [16], modified by Kisworo and Depamede [17] and Nurhaerani et al. [18]. ...
This study aimed to isolate IgG from chicken serum using a combination of coconut caprylic triglyceride and ammonium sulfate precipitation techniques. This study used serum from chickens that had previously been vaccinated repeatedly using a commercial rabies vaccine. Initially, the serum was treated with 2.5% coconut caprylic triglyceride, followed by serum IgG precipitation using 40% ammonium sulfate. The 10% SDS-PAGE analysis showed that a protein of about 190 kDa with a purity above 95% was successfully isolated. Whether the isolate protein is IgG and specific to rabies antigen still needs to be investigated further.
... SDS-PAGE was carried out following Laemmli's protocol [19] with slight modifications. About 10-20 μL of crude extracts, precipitated samples, and dialysate were injected separately into the electrophoresis gel. ...
Trypsin production from skipjack tuna (Katsuwonus pelamis) viscera is one significant way to increase the value of fish’s industrial waste. The present work reports the biochemical properties of trypsin from skipjack tuna viscera. The trypsin was fractionated using 0–60% ammonium sulfate and dialyzed. The enzyme was characterized to find the optimum temperature and pH for the substrate N-α-benzoyl-dl-arginine-p-nitroanilide. The 40–50% ammonium sulfate fractionation showed the highest activity at a specific activity of 1.66 U/mg and yield of 69.91%. Specific activity increased after dialysis to 2.17 U/mg with 4.49 times purity and yield of 39.20%. The molecular weights of the enzymes were estimated as 25, 29, and 35 kDa based on the enzyme activity separated by electrophoresis. The enzyme worked optimally at a temperature and pH of 50–60°C and 8.0, respectively. Metal ions (Ca²⁺, K⁺, Na⁺, Mg²⁺) at a concentration of 20 mM showed no influence on the activity. Enzyme activity was inhibited by Zn²⁺ at 20 mM, phenyl methyl sulfonyl fluoride (PMSF), benzamidine, and soybean trypsin inhibitor (SBTI), which confirmed the characteristics of a serine protease.
... Phosphorylation of PKA substrates PKA substrate phosphorylation was assessed as previously reported 37 . After capacitation, sperm suspensions were washed, resuspended in the Laemmli sample buffer 49 , boiled and centrifuged. Supernatants were boiled again in the presence of 70 mM 2-β-mercaptoethanol. Proteins (corresponding to 3 × 10 6 sperm/lane) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. ...
To acquire the ability to fertilize the egg, mammalian spermatozoa must undergo a series of changes occurring within the highly synchronized and specialized environment of the female reproductive tract, collectively known as capacitation. In an attempt to replicate this process in vitro, various culture media for mouse sperm were formulated over the past decades, sharing a similar overall composition but differing mainly in ion concentrations and metabolic substrates. The widespread use of the different media to study the mechanisms of capacitation might hinder a comprehensive understanding of this process, as the medium could become a confounding variable in the analysis. In this context, the present side-by-side study compares the influence of four commonly used culture media (FD, HTF and two TYH versions) on mouse sperm capacitation. We evaluated the induction of protein kinase A phosphorylation pathway, motility, hyperactivation and acrosome reaction. Additionally, in vitro fertilization and embryo development were also assessed. By analyzing these outcomes in two mouse colonies with different reproductive performance, our study provides critical insights to improve the global understanding of sperm function. The results obtained highlight the importance of considering variations in medium composition, and their potential implications for the future interpretation of results.
... SDS-PAGE was performed utilizing the method of Leammli (Laemmli, 1970). A sample buffer containing 2% SDS and bromophenol was added to each fraction after column chromatography. ...
Scallops are one of the main marine products of Hokkaido, Japan. In addition to adductor muscle, scallop mantle tissue is often consumed in Japan. Previously, we showed that feeding mice a diet containing 1% mantle tissue resulted in lower food consumption and, ultimately, death. In this study, we isolated and identified toxic substances from scallop mantle tissue. The isolated toxic substances were protein complexes with molecular weights of 18 kDa and 29 kDa. Feeding mice a diet containing 0.05% toxic substances led to their death at five weeks. Based on LC-MS/MS analysis, the 29-kDa and 18-kDa proteins were identified as an actin fragment and the N-terminal fragment of the gelsolin-like protein, respectively. The 18-kDa protein was expressed in the mantle, gill, and ovary but not in the adductor muscle, testis, or midgland. Toxicity was observed only in mouse tissues expressing the 18-kDa protein. Feeding mice a diet containing only the 18-kDa protein did not induce decreased food consumption or death, implying that both the 29-kDa and 18-kDa complexes are essential for toxicity. This is the first study to identify a novel toxin in scallop tissues.
... The obtained fractions tested for lipase activity were further subjected to SDS-PAGE to ensure the homogeneity and to determine molecular weight [16]. The protein bands were observed after staining the gel with Coomassie brilliant blue R-250. ...
Polyhydroxyalkanoates (PHAs) constitute a principal group of biodegradable polymers that are produced by certain microbes under limited supply of nutrients. PHA is a linear polyester that comprises of 3-hydroxy fatty acid monomers. Triacylglycerol acylhydrolases are known to catalyze the hydrolysis of ester linkages and in turn they are beneficial in the degradation of PHA. In present study, lipase-catalyzed degradation of PHA synthesized by Priestia megatarium POD1 was monitored. A gene from thermotolerant Bacillus subtilis TTP-06 that was capable of expressing lipase enzyme was amplified by PCR, cloned into a pTZ57R/T-vector, transferred to an expression vector pET-23a (+) and expressed in Escherichia coli BL21 (DE3) cells. The recombinant enzyme purified to 19.37-fold had a molecular weight of 30 kDa (SDS-PAGE analysis). Scanning Electron Microscopy (SEM) revealed changes in the surface morphology of native and treated PHA films. Further, changes in molecular vibrations were confirmed by Fourier Transform Infrared Spectroscopy.
... The SDS-PAGE method 24 was used to determine the molecular weights of proteins obtained under optimum extraction conditions. In the study, 12% gel was used and 10 µg and 50 µg of samples were loaded. ...
The study aims to optimize the extraction process and characterize the proteins found in fenugreek seeds. The water and oil holding capacities, coagulated protein content, foaming, and emulsification properties of the isolated proteins were investigated under all extraction conditions. Also, solubility, molecular weights, structural and thermal properties were determined. In the extraction processes carried out at different pH (pH 6.0–12.0) and solid:solvent ratios (20–60 g/L), it was determined that the highest extraction yield (94.3 ± 0.3%) was achieved when the pH was 11.47 and the solid-solvent ratio was 34.50 g/L. Three distinct bands (46, 59, and 80 kDa) in the range of 22–175 kDa were determined for the fenugreek seed protein isolate obtained under optimum extraction conditions. Protein secondary structures were determined using Fourier Transform Infrared (FT-IR) spectra and it was determined that β-sheet structures were highly present. In addition, denaturation temperature and denaturation enthalpy were calculated as ~119 °C and 28 mJ/g, respectively.
... Protein electrophoresis was carried out according to Laemmli [46] in a Mini Protean 3 system (Bio-Rad). Resolving and stacking gels were prepared at 12% w/v and 5% w/v polyacrylamide, respectively. ...
Follicle-stimulating hormone (FSH) is an important protein used for bovine ovarian hyperstimulation in multiple ovulation and embryo transfer technology (MOET). Several attempts to produce bovine FSH (bFSH) in recombinant systems have been reported, nonetheless, up to date, the most commonly used products are partially purified preparations derived from porcine or ovine (pFSH or oFSH) pituitaries. Here we describe the development of a biotechnology process to produce a novel, hyperglycosylated, long-acting recombinant bFSH (LA-rbFSH) by fusing copies of a highly O-glycosylated peptide. LA-rbFSH and a nonmodified version (rbFSH) were produced in suspension CHO cell cultures and purified by IMAC with high purity levels (>99%). LA-rbFSH presented a higher glycosylation degree and sialic acid content than rbFSH. It also demonstrated a notable improvement in pharmacokinetic properties after administration to rats, including a higher concentration in plasma and a significant (seven-fold) reduction in apparent clearance (CLapp). In addition, the in vivo specific bioactivity of LA-rbFSH in rats was 2.4-fold higher compared to rbFSH. These results postulate this new molecule as an attractive substitute for commercially available porcine pituitary-derived products.
... A caracterização das proteínas e oligopeptídeos obtidos depois da hidrólise, foi realizada uma eletroforese com gél de poliacrilamida (Tris-Tricina), conforme metodologia de Laemmli (1970). ...
Os tubérculos do gênero Dioscorea tem relevante importância socioeconômica para a região Nordeste do Brasil, o inhame da costa (Dioscorea cayennensis) é uma hortaliça que apresenta ótimos valores nutricionais e energéticos, importante na dieta da população, já o cará-moela (Dioscorea bulbifera) é uma espécie de limitada comercialização e de pouco conhecimento pelos brasileiros, com familiarização entre curandeiros e donas de casa, que o usam para fins medicinais. Há várias pesquisas envolvendo o gênero que evidenciam os interesses em explorar seus potenciais, por isso, o objetivo deste trabalho é comparar a atividade antioxidantes os hidrolisados obtidos por ação da enzima pepsina, das proteínas biologicamente ativas presentes em tubérculos de D. cayannensis e D. bulbifera e caracterizar o perfil de seus peptídeos bioativos. Assim, fez-se necessário produzir uma farinha fina que foi submetida a extração das proteínas em tampão glicina 0,1 mol/L e em água ultra pura, ambos em pH 9. Determinado o teor de proteínas solúveis pelo método de Bradford (1976) que demonstrou que as proteínas de D. bulbifera se solubiliza melhor em solução tampão de Gly e D. cayannensis em H2O. Os concentrados proteícos foram submetidos à hidrólise com pepsina, e os peptídeos formados foram confirmados em eletroforese de poliacrilamida (Tris-Tricina). A atividade antioxidante foi realizada com o radical de ABTS+ e os resultados constataram que houve relevância do grau de hidrólise das proteínas pela enzima pepsina em relação aos concentrados proteicos, em algumas amostras, e o inhame teve maior capacidade de sequestro do radical se comparado ao cará-moela. Pesquisas como essas agregam valores e contribui para valorização e expansão do conhecimento destas espécies de tubérculos cultivadas e consumidas na região.
... SDS-PAGE was carried out according to the procedure of Laemmli (1970) with a discontinuous buffer system (Garfin 2009). Briefly, 25 μg of protein was loaded onto each well of 0.75 mm-thick gel. ...
Microbes play an essential role in soil fertility by replenishing the nutrients; they encounter various biotic and abiotic stresses disrupting their cellular homeostasis, which expedites activating a conserved signaling pathway for transient over-expression of heat shock proteins (HSPs). In the present study, a versatile soil bacterium Bacillus subtilis strain PSK.A2 was isolated and characterized. Further, the isolated bacterium was exposed with several stresses, viz., heat, salt, acid, alkaline, and antibiotics. Stress-attributed cellular morphological modifications such as swelling, shrinkage, and clump formation were observed under the scanning electron microscope. The comparative protein expression pattern was studied by SDS-PAGE, relative protein stabilization was assessed by protein aggregation assay, and relative survival was mapped by single spot dilution and colony-counting method under control, stressed, lethal, and stressed lethal conditions of the isolate. The findings demonstrated that bacterial stress tolerance was maintained via the activation of various HSPs of molecular weight ranging from 17 to 115 kD to respective stimuli. The treatment of subinhibitory dose of antibiotics not interfering protein synthesis (amoxicillin and ciprofloxacin) resulted in the expression of eight HSPs of molecular weight ranging from 18 to 71 kD. The pre-treatment of short stress dosage showed endured overall tolerance of bacterium to lethal conditions, as evidenced by moderately enhanced total soluble intracellular protein content, better protein stabilization, comparatively over-expressed HSPs, and relatively enhanced cell survival. These findings hold an opportunity for developing novel approaches towards enhancing microbial resilience in a variety of conditions, including industrial bioprocessing, environmental remediation, and infectious disease management.
... Characterization of SPIE n Protein gel electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted by using a separating gel with a concentration of 15% and a stacking gel with a concentration of 5% in accordance with a previously described procedure 25 with some modifications. For this, 10 μL of sample (protein concentration, 2.0 g L −1 ) was combined with 10 μL of 1× loading buffer and heat-treated in hot water for 10 min. ...
BACKGROUND
Recent studies have shown that the wettability of protein‐based emulsifiers is critical for emulsion stability. However, few studies have been conducted to investigate the effects of varying epigallocatechin gallate (EGCG) concentrations on the wettability of protein‐based emulsifiers. Additionally, limited studies have examined the effectiveness of soy protein–EGCG covalent complex nanoparticles with improved wettability as emulsifiers for stabilizing high‐oil‐phase (≥ 30%) curcumin emulsions.
RESULTS
Soy protein isolate (SPI)–EGCG complex nanoparticles (SPIEn) with improved wettability were fabricated to stabilize high‐oil‐phase curcumin emulsions. The results showed that EGCG forms covalent bonds with SPI, which changes its secondary structure, enhances its surface charge, and improves its wettability. Moreover, SPIEn with 2.0 g L ⁻¹ EGCG (SPIEn‐2.0) exhibited a better three‐phase contact angle (56.8 ± 0.3o) and zeta potential (−27 mV) than SPI. SPIEn‐2.0 also facilitated the development of curcumin emulsion gels at an oil volume fraction of 0.5. Specifically, the enhanced network between droplets as a result of the packing effects and SPIEn‐2.0 with inherent antioxidant function was more effective at inhibiting curcumin degradation during long‐term storage and ultraviolet light exposure.
CONCLUSION
The results of the present study indicate that SPIEn with 2.0 g L ⁻¹ EGCG (SPIEn‐2.0) comprises the optimum conditions for fabricating emulsifiers with improved wettability. Additionally, SPIEn‐0.2 can improve the physicochemical stability of high‐oil‐phase curcumin emulsions, suggesting a novel strategy to design and fabricate high‐oil‐phase emulsion for encapsulating bioactive compounds. © 2024 Society of Chemical Industry.
... The eluted intravenous immunoglobulins were analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-SDS-PAGE) using 5% stacking gel and 12% resolving gel, according to the Laemmli method with modifications [81]. The samples were dissolved in a Laemmli sample buffer containing 10 mM DTT as the reducing agent. ...
The discovery and investigation of new natural compounds with antimicrobial activity are new potential strategies to reduce the spread of antimicrobial resistance. The presented study reveals, for the first time, the promising antibacterial potential of two fractions from Cornu aspersum mucus with an MW < 20 kDa and an MW > 20 kDa against five bacterial pathogens—Bacillus cereus 1085, Propionibacterium acnes 1897, Salmonella enterica 8691, Enterococcus faecalis 3915, and Enterococcus faecium 8754. Using de novo sequencing, 16 novel peptides with potential antibacterial activity were identified in a fraction with an MW < 20 kDa. Some bioactive compounds in a mucus fraction with an MW > 20 kDa were determined via a proteomic analysis on 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and bioinformatics. High homology with proteins and glycoproteins was found, with potential antibacterial activity in mucus proteins named aspernin, hemocyanins, H-lectins, and L-amino acid oxidase-like protein, as well as mucins (mucin-5AC, mucin-5B, mucin-2, and mucin-17). We hypothesize that the synergy between the bioactive components determined in the composition of the fraction > 20 kDa are responsible for the high antibacterial activity against the tested pathogens in concentrations between 32 and 128 µg/mL, which is comparable to vancomycin, but without cytotoxic effects on model eukaryotic cells of Saccharomyces cerevisiae. Additionally, a positive effect, by reducing the levels of intracellular oxidative damage and increasing antioxidant capacity, on S. cerevisiae cells was found for both mucus extract fractions of C. aspersum. These findings may serve as a basis for further studies to develop a new antibacterial agent preventing the development of antibiotic resistance.
... Lysates were transferred to a new 1.5 mL tube and beads were washed three times with 5 % TCA. Precipitated protein was collected at 6,000 RPM in an Eppendorf centrifuge for 10 minutes, resuspended in 1.5 x Laemmli sample buffer (Laemmli, 1970), and neutralized with one-third volume of Tris-base, before analysis by gel electrophoresis. Immunoblots were conducted as described (Bashkirov et al., 2000). ...
Stalled replication forks can be processed by several distinct mechanisms collectively called post-replication repair which includes homologous recombination, fork regression, and translesion DNA synthesis. However, the regulation of the usage between these pathways is not fully understood. The Rad51 protein plays a pivotal role in maintaining genomic stability through its roles in HR and in protecting stalled replication forks from degradation. We report the isolation of separation-of-function mutations in Saccharomyces cerevisiae Rad51 that retain their recombination function but display a defect in fork protection leading to a shift in post-replication repair pathway usage from HR to alternate pathways including mutagenic translesion synthesis. Rad51-E135D and Rad51-K305N show normal in vivo and in vitro recombination despite changes in their DNA binding profiles, in particular to dsDNA, with a resulting effect on their ATPase activities. The mutants lead to a defect in Rad51 recruitment to stalled forks in vivo as well as a defect in the protection of dsDNA from degradation by Dna2-Sgs1 and Exo1 in vitro . A high-resolution cryo-electron microscopy structure of the Rad51-ssDNA filament at 2.4 Å resolution provides a structural basis for a mechanistic understanding of the mutant phenotypes. Together, the evidence suggests a model in which Rad51 binding to duplex DNA is critical to control pathway usage at stalled replication forks.
... Sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a RAPIDAS AE-6500 apparatus (ATTO, Tokyo, Japan) with 1-mm thick precast slab gels containing 10%-20% gradient acrylamide (e-PAGEL E-T1020L; ATTO), according to the procedure described by Laemmli (1970). 100 μg of each protein extract (Pp, Ps, and H) were loaded onto the gel. ...
Introduction
Several fluorescent proteins (FPs) and chromoproteins (CPs) are present in anthozoans and play possible roles in photoprotection. Coral tissues in massive corals often display discoloration accompanied by inflammation. Incidences of the pink pigmentation response (PPR) in massive Porites , described as inflammatory pink lesions of different shapes and sizes, has recently increased worldwide. FPs are reported to be present in PPR lesions, wherein a red fluorescent protein (RFP) appears to play a role in reducing reactive oxygen species. However, to date, the biochemical characterization and possible roles of the pigments involved are poorly understood. The present study aimed to identify and characterize the proteins responsible for pink discoloration in massive Porites colonies displaying PPRs, as well as to assess the differential distribution of pigments and the antioxidant properties of pigmented areas.
Method
CPs were extracted from PPR lesions using gel-filtration chromatography and identified via genetic analysis using liquid chromatography-tandem mass spectrometry. The coexistence of CPs and RFP in coral tissues was assessed using microscopic observation. Photosynthetic antivity and hydrogen peroxide-scavenging activitiy were measured to assess coral stress conditions.
Results
The present study revealed that the same CP (plut2.m8.16902.m1) isolated from massive Porites was present in both the pink spot and patch morphologies of the PPR. CPs were also found to coexist with RFP in coral tissues that manifested a PPR, with a differential distribution (coenosarc or tip of polyps’ tentacles). High hydrogen peroxide-scavenging rates were found in tissues affected by PPR.
Discussion and Conclusion
The coexistence of CPs and RFP suggests their possible differential role in coral immunity. CPs, which are specifically expressed in PPR lesions, may serve as an antioxidant in the affected coral tissue. Overall, this study provides new knowledge to our understanding of the role of CPs in coral immunity.
... The determination of protein molecular weights in the fenugreek seed protein isolate obtained under optimum conditions was carried out using the SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) method (Laemmli, 1970 (10 μg) were cut, spliced, and re-colored for figure construction. ...
In this study, fenugreek seed proteins were extracted using ultrasonic‐assisted extraction with varying solid:solvent ratios (20–60 g/L) and sonication amplitudes (30%–80%) to determine optimal conditions for the highest extraction yield. The functional, structural, and nutritional characteristics of the protein isolates of fenugreek seeds were investigated. The highest yield (98.74 ± 0.49%) was achieved at a solid:solvent ratio of 43.83 g/L and an amplitude of 67.51%. The coagulated protein values of fenugreek seed protein isolates ranged from ~15.8% to 31.2%, water‐holding capacities ranged from ~2.2 to 3.2 g/g, oil‐holding capacities ranged from ~2.6 to 4.1 g/g, foaming capacities ranged from ~16.3% to 21.3%, foam stabilities ranged from ~59.7% to 78.1%, emulsion stabilities ranged from ~30.2 to 34.5 min, emulsion activities ranged from ~73.8 to 76.8 m²/g, and emulsion capacities ranged from ~26.9% to 30.5% under different extraction conditions. SDS‐PAGE analysis revealed three distinct bands (46, 59, and 80 kDa) for the protein isolates. FT‐IR spectroscopy showed a high presence of β‐sheet structures. The amino acid composition analysis of fenugreek seed protein isolates was determined, revealing richness in essential amino acids (317.97 g amino acid/kg protein isolate). In addition, cupcakes enriched with protein isolates (5%, 10%, and 20% as flour substitutes) were produced, and quality properties such as color change, browning index, moisture content, water activity, baking yield, bulk density, hardness, volume, symmetry, and uniformity indexes were determined. The application of protein isolates in cupcake production demonstrated the potential of fenugreek seeds as valuable ingredients for enhancing the nutritional profile of bakery products.
... The protein was eluted using a linear gradient of buffer A containing 500 mM imidazole in an Akta Purifier (Cytiva). The molecular mass and purity of TfEst were checked by SDS-PAGE under denaturing conditions [14] and the protein concentration was measured by the spectral method described in [15]. ...
Background
Esterases (EC 3.1.1.X) are enzymes that catalyze the hydrolysis ester bonds. These enzymes have large potential for diverse applications in fine industries, particularly in pharmaceuticals, cosmetics, and bioethanol production.
Methods and results
In this study, a gene encoding an esterase from Thermobifida fusca YX (TfEst) was successfully cloned, and its product was overexpressed in Escherichia coli and purified using affinity chromatography. The TfEst kinetic assay revealed catalytic efficiencies of 0.58 s⁻¹ mM⁻¹, 1.09 s⁻¹ mM⁻¹, and 0.062 s⁻¹ mM⁻¹ against p-Nitrophenyl acetate, p-Nitrophenyl butyrate, and 1-naphthyl acetate substrates, respectively. Furthermore, TfEst also exhibited activity in a pH range from 6.0 to 10.0, with maximum activity at pH 8.0. The enzyme demonstrated a half-life of 20 min at 70 °C. Notably, TfEst displayed acetyl xylan esterase activity as evidenced by the acetylated xylan assay. The structural prediction of TfEst using AlphaFold indicated that has an α/β-hydrolase fold, which is consistent with other esterases.
Conclusions
The enzyme stability over a broad pH range and its activity at elevated temperatures make it an appealing candidate for industrial processes. Overall, TfEst emerges as a promising enzymatic tool with significant implications for the advancement of biotechnology and biofuels industries.
... Total proteins were extracted from different leaves of transplastomic and non-transformed (NT) tobacco plants. Leaf tissue (25 mg) was manually grinded in 125 µL of Laemmli buffer 1X (Laemmli 1970) and heated at 98 °C for 10 min. The homogenate was centrifuged for 10 min at 16,000 g and 20 µL of the remaining supernatant was loaded in a polyacrylamide gel and electrophoresed for protein separation and further western blot analysis. ...
Main conclusion
We generated transplastomic tobacco lines that stably express a human Basic Fibroblast Growth Factor (hFGFb) in their chloroplasts stroma and purified a biologically active recombinant hFGFb.
Main
The use of plants as biofactories presents as an attractive technology with the potential to efficiently produce high-value human recombinant proteins in a cost-effective manner. Plastid genome transformation stands out for its possibility to accumulate recombinant proteins at elevated levels. Of particular interest are recombinant growth factors, given their applications in animal cell culture and regenerative medicine. In this study, we produced recombinant human Fibroblast Growth Factor (rhFGFb), a crucial protein required for animal cell culture, in tobacco chloroplasts. We successfully generated two independent transplastomic lines that are homoplasmic and accumulate rhFGFb in their leaves. Furthermore, the produced rhFGFb demonstrated its biological activity by inducing proliferation in HEK293T cell lines. These results collectively underscore plastid genome transformation as a promising plant-based bioreactor for rhFGFb production.
... The homogenate was centrifuged at 16,000× g at 4 °C for 20 min. The resulting supernatant was subjected to native polyacrylamide gel electrophoresis (PAGE) on 7.5% separating gels in a Laemmli buffer system without SDS [76]. After electrophoresis, AO activity was stained as previously described [18] using indole-3-aldehyde as substrate. ...
Abscisic acid (ABA) plays a crucial role in plant defense mechanisms under adverse environmental conditions, but its metabolism and perception in response to heavy metals are largely unknown. In Pisum sativum exposed to CdCl2, an accumulation of free ABA was detected in leaves at different developmental stages (A, youngest, unexpanded; B1, youngest, fully expanded; B2, mature; C, old), with the highest content found in A and B1 leaves. In turn, the content of ABA conjugates, which was highest in B2 and C leaves under control conditions, increased only in A leaves and decreased in leaves of later developmental stages after Cd treatment. Based on the expression of PsNCED2, PsNCED3 (9-cis-epoxycarotenoid dioxygenase), PsAO3 (aldehyde oxidase) and PsABAUGT1 (ABA-UDP-glucosyltransferase), and the activity of PsAOγ, B2 and C leaves were found to be the main sites of Cd-induced de novo synthesis of ABA from carotenoids and ABA conjugation with glucose. In turn, β-glucosidase activity and the expression of genes encoding ABA receptors (PsPYL2, PsPYL4, PsPYL8, PsPYL9) suggest that in A and B1 leaves, Cd-induced release of ABA from inactive ABA-glucosyl esters and enhanced ABA perception comes to the forefront when dealing with Cd toxicity. The distinct role of leaves at different developmental stages in defense against the harmful effects of Cd is discussed.
BACKGROUND
Cellobiose 2‐epimerase (CE) has received great attention due to its potential applications in the food and pharmaceutical industries. In this study, a novel CE from mesophilic anaerobic halophilic bacterium Iocasia fonsfrigidae strain SP3‐1 (IfCE) was successfully expressed in Escherichia coli and characterized.
RESULTS
Unlike other CEs, the purified IfCE shows only epimerization activity toward β‐1,4‐glycosidic linkages of disaccharides, including mannobiose, cellobiose and lactose, but not for monosaccharides, β‐1,4‐glycosidic linkages of trisaccharides and α‐1,4‐glycosidic linkages of disaccharides. Only one epimerization product was obtained from the action of IfCE against mannobiose, cellobiose and lactose. Under optimum conditions, 31.0% of epilactose, a rare and low‐calorie prebiotic sweetener with medicinal and pharmacological properties, was obtained from 10 mg mL⁻¹ lactose. IfCE was highly active against lactose under NaCl concentrations up to 500 mmol L⁻¹, possibly due to the excessive basic (arginine and lysine) and acidic (aspartic and glutamic acids) amino acid residues, which are localized on the surface of the halophilic enzyme structure. These residues may protect the enzyme from Cl⁻ and Na⁺ ions from the environment, respectively. Under normal conditions, IfCE was able to convert lactose present in fresh goat milk to epilactose with a conversion yield of 31% in 10 min. In addition, IfCE has been investigated as a safe enzyme for human allergen.
CONCLUSION
The results suggested that IfCE is a promising candidate to increase the quality and value of milk and dairy products by converting lactose that causes digestive problems in people with lactose intolerance into epilactose. © 2024 Society of Chemical Industry.
Autoantibodies against apolipoprotein A-I (ApoA-I) are associated with cardiovascular disease risks. We aimed to examine the 4-hydroxy-2-nonenal (HNE) modification of ApoA-I in coronary artery disease (CAD) and evaluate the potential risk of autoantibodies against their unmodified and HNE-modified peptides. We assessed plasma levels of ApoA-I, HNE-protein adducts, and autoantibodies against unmodified and HNE-peptide adducts, and significant correlations and odds ratios (ORs) were examined. Two novel CAD-specific HNE-peptide adducts, ApoA-I251–262 and ApoA-I70–83, were identified. Notably, immunoglobulin G (IgG) anti-ApoA-I251–262 HNE, IgM anti-ApoA-I70–83 HNE, IgG anti-ApoA-I251–262, IgG anti-ApoA-I70–83, and HNE-protein adducts were significantly correlated with triglycerides, creatinine, or high-density lipoprotein in CAD with various degrees of stenosis (<30% or >70%). The HNE-protein adduct (OR = 2.208-fold, p = 0.020) and IgM anti-ApoA-I251–262 HNE (2.046-fold, p = 0.035) showed an increased risk of progression from >30% stenosis in CAD. HNE-protein adducts and IgM anti-ApoA-I251–262 HNE may increase the severity of CAD at high and low levels, respectively.
Previously, we showed that water extract (soymilk, except pH was increased to 8 from 6.5) of whole soybean could be used directly as a raw material for producing edible soy films by deposition of the film‐forming solution (soy extract with enhancers). However, the strength of such soy films needed improvement because they were weak. The purpose of this study was to investigate how transglutaminase (TG) cross‐linking reactions and film enhancers, including pectin (low‐ and high‐methoxyl pectin), whey protein isolate (WPI), and soy protein isolate (SPI), improve the physical properties of soy films. Soy films prepared with TG had tensile strength (TS) of 3.01 MPa and puncture strength (PS) of 0.78 MPa, which were higher by as much as 51% and 30% than that of soy films without TG treatment, respectively. Pectin showed significant effects on the mechanical properties of TG‐added soy films in terms of TS, PS, and % elongation. On the other hand, only TS and PS were increased by the addition of WPI or SPI. Heat curing had a significant effect on soy film's physical properties. TG treatment significantly reduced film solubility when soaked in water and various levels of acid (vinegar) and base (baking soda) solutions. Under the experimental conditions of 35 unit TG and 28 min of reaction, the degrees of cross‐linking were evidenced by the disappearance of individual protein subunits, except the basic subunit of glycinin, and the reduction of 21% of lysine residues of the proteins.
Highlights
Edible soy films were made with transglutaminase and about 21% lysine cross‐linked.
The mechanical strength of soy films was increased by incorporating film enhancers.
Transglutaminase enhanced the mechanical properties of soy films.
In this laboratory exercise, sarcoplasmic muscle proteins of fish are extracted with a salt solution, the protein content of the extract is measured by the bicinchoninic acid (BCA) colorimetric assay, and the proteins in the fish extracts are separated and visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Visualization of the protein banding patterns (subunit size and relative quantity) makes it possible to distinguish among different types of fish since many fish have a characteristic protein subunit pattern.
Objective: The aim in this study was to analyze the solubility and immunoglobin E (IgE) -binding ability of the egg white proteins in boiled eggs during boiling.
Methods: Boiled eggs were prepared by cooking large commercial hen's eggs in boiling water. Protein extraction from the raw (boiled for 0 min) and boiled egg whites, after lyophilization, was performed with phosphate-buffered saline (PBS), sodium dodecyl sulfate and urea solution, and 2-mercaptoethanol (2-ME) solution; the extracts were analyzed using the inhibition ELISA, SDS-PAGE, and IgE immunoblotting.
Results: The amount of ovalbumin (OVA) in the PBS fraction decreased significantly over time during 7 and 9 minutes of boiling. The SDS-PAGE results showed that the ovotransferrin, OVA, and lysozyme disappeared over time in the PBS fraction but were detected in the 2-ME solution fraction. Additionally, the band presumed to be ovomucoid (OVM) did not disappear from the PBS fraction after 20 minutes of boiling. The bands of IgE-binding proteins in the PBS fraction decreased at approximately 5 and 10 minutes.
Conclusions: Egg white proteins except OVM gradually became insoluble in water up to 10 minutes of boiling time and did not change thereafter. The results of this study provide basic data on the guidance of heated egg consumption.
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Background
Tuberculosis (TB), a global infectious threat, has seen a concerning rise in aminoglycoside-resistant Mycobacterium tuberculosis (M.tb) strains. The potential role of capsule proteins remains largely unexplored. This layer acts as the primary barrier for tubercle bacilli, attempting to infiltrate host cells and subsequent disease development.
Methods
The study aims to bridge this gap by investigating the differentially expressed capsule proteins in aminoglycoside-resistant M.tb clinical isolates compared with drug-sensitive isolates employing two-dimensional gel electrophoresis, mass spectrometry, and bioinformatic approaches.
Results
We identified eight proteins that exhibited significant upregulation in aminoglycoside-resistant isolates. Protein Rv3029c and Rv2110c were associated with intermediary metabolism and respiration; Rv2462c with cell wall and cell processes; Rv3804c with lipid metabolism; Rv2416c and Rv2623 with virulence and detoxification/adaptation; Rv0020c with regulatory functions; and Rv0639 with information pathways. Notably, the Group-based Prediction System for Prokaryotic Ubiquitin-like Protein (GPS-PUP) algorithm identified potential pupylation sites within all proteins except Rv3804c. Interactome analysis using the STRING 12.0 database revealed potential interactive partners for these proteins, suggesting their involvement in aminoglycoside resistance. Molecular docking studies revealed suitable binding between amikacin and kanamycin drugs with Rv2462c, Rv3804c, and Rv2623 proteins.
Conclusion
As a result, our findings illustrate the multifaceted nature of aminoglycoside resistance in M.tb and the importance of understanding how capsule proteins play a role in counteracting drug efficacy. Identifying the role of these proteins in drug resistance is crucial for developing more effective treatments and diagnostics for TB.
Background
Phosphorus (P) is one of the nonrenewable resources of critical importance in agricultural production. P is present in soil in organic and inorganic forms. Phytate constitutes the majority of organic P in soil. Phytate binds strongly to the solid phase of the soil and becomes unavailable for use by plants. Therefore, the soluble phytate‐P ratio in soil is mostly at very low levels. Plants and associated microorganisms secrete organic acids and hydrolyzing enzymes such as phytase to dissolve phytate in the soil. Both the solubility of phytate and phytase activity are limiting properties for the uptake of phytate‐P by plants.
Aims
Our aim was to evaluate the effects of phytase immobilized on zinc oxide nanoparticles (ZnO Np) on tomato plant ( Solanum lycopersicum ) growth parameters. In this study, seedling period was analyzed.
Methods
In the study, phytase activity of 13 different bacteria was investigated, and phytase was purified from Lactobacillus kefiri , showing the highest activity, and its biochemical properties were determined. Phytase was immobilized on zinc oxide (ZnO) nanoparticles and characterized by X‐ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopes analysis. The effects of ZnONps, immobilized phytase, and free phytase on the growth parameters of tomato plant were investigated. Tomato seeds were soaked with ZnONps, immobilized and free phytase for 30 min at room temperature and sown in pots containing suitable growing medium. Vegetative development of tomato plant, plant height, number of lateral branches, main stem diameter, distance between nodes, number of nodes, main root, and shoot length were determined.
Results
Phytase was partially purified with 7.60% recovery and specific activity of 1758.5 (EU mg ⁻¹ protein). Molecular mass of partially purified phytase was approx.72 kD, optimum pH and temperature values were determined as pH 5.0 and 70–80°C, respectively. Immobilized phytase caused a significant increase of 41.1% in plant height, 64.1% in main root, and 36.1% in shoot length in tomato plants compared to the control. In addition, a significant increase was observed in the number of side branches, main stem diameter, distance between nodes, number of nodes, and vegetative growth of the plant.
Conclusions
The results showed that the immobilized phytase enzyme has a positive effect on seedling growth in tomato and can be used in tomato cultivation in the future.
Brasilicardin A, BraA, is a secondary metabolite produced by the bacterium Nocardia terpenica , and a promising drug due to its potent immunosuppressive activity and low cytotoxicity. Currently, a semisynthetic approach confers production of a complete compound but suffers from insufficient heterologous biosynthesis of BraA intermediates used in the chemical semi-synthesis steps leading to only lab scale quantities of the compound. A better understanding of the involved gene expression regulatory pathways within the brasilicardin biosynthetic gene cluster, Bra-BGC, is a prerequisite to further improve production titers. However, the transcriptional regulation of the Bra-BGC has only been superficially analyzed, till now.
In this study, we comprehensively analyze the functions of several unstudied transcriptional regulators, KstR, SdpR and OmpR, encoded within the close vicinity of the Bra-BGC, and delve into the role of the previously described cluster-situated activator Bra12. We present, that Bra12 and the novel regulator SdpR, bind several DNA sequences located in the promoter regions of the genes essential for BraA biosynthesis. Subsequently, we demonstrate the complex regulatory network through which both regulators are capable of controlling activity of those gene promoters and thus gene expression in Bra-BGC. Furthermore, using the heterologous producer strain Amycolatopsis japonicum , we present, that Bra12 and SdpR regulators play opposite roles in brasilicardin congener biosynthesis. Finally, we propose a comprehensive model of multilevel gene expression regulation in Bra-BGC and propose the roles of locally encoded transcriptional regulators.
Engineering keratin plastics from chicken feather wastes is highly attractive from an environmental prospect but challenging due to the influence of extraction routes on the macromolecular keratin features. In this study, the effect of precipitation methods on desired properties of biodegradable keratin films was investigated. Keratin was extracted from an industrial chicken feather waste by sulfitolysis, and further recovered using hydrochloric acid (KHA), citric acid (KCA), sodium chloride (KSC), and acetone (KAT) as precipitating agents. Keratin films containing glycerol at 25 wt% were produced by compression molding. All keratin films were nearly colorless and highly transparent to visible light while exhibiting good UV barrier properties. The KAT and KSC films showed greater ductility ( ε break > 50%) compared with KHA and KCA, which was correlated with broader molecular weight distributions and less crosslinking in the former keratins. Importantly, disulfide bonds created in the processing led to keratin films with low water solubility. Larger surface hydrophobicity was attained from keratin recovered by organic solvent precipitation (KAT). These results advance the understanding of precipitating agents as physicochemical property modifiers of biodegradable keratin films for food packaging and other applications.
The product of gene 31 (P31) of bacteriophage T4 is required for the formation of the phage capsid and its related structures. In the absence of active P31, product P23, the major component of the phage capsid, aggregates into “lumps” which sediment with the cell envelope. Temperature-shift experiments with ts-mutants in gene 31 demonstrate that the P23 aggregates can be dissolved by activated P31 and the dissolved P23 is normal, in that it can be used for incorporation into active phage. It is possible that P31 acts catalytically.Two different ts-mutants in gene 31 produce two different temperature-sensitive proteins. One is irreversibly inactivated if produced at the restrictive temperature; but when synthesized at the permissive temperature, it becomes heat stable and remains functional at the restrictive temperature. The other is reversibly affected by temperature, activated following shift to permissive temperature and inactivated if restrictive temperature is established.
Two series of experiments were performed, utilizing a modification of the hemolysin plaque technique which registers 19S antibody, in an attempt to determine the frequency of cells capable of simultaneously producing antibody to two non-cross-reacting antigens. Mice were immunized i.v. with rabbit and camel RBC and their spleens assayed for cells producing antibody against both antigens. 16,904 cells producing antibody of one or the other specificity, from 26 mice, were counted. Not one cell was detected which produced antibody of two specificities. Rabbits were immunized intradermally with HSA to which polyalanyl and p-azobenzenearsonate groups were chemically attached. The individual haptens, polyalanyl, and p-azobenzenearsonate groups were coupled to separate aliquots of SRBC, and the lymph nodes of immunized rabbits were assayed for cells releasing antibody against both haptens. In a study of 11 rabbits, after counting 27,845 cells producing antibody, we detected no "double" plaques.
Forty proteins with polypeptide chains of well characterized molecular weights have been studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate following the procedure of Shapiro, Viñuela, and Maizel (Biochem. Biophys. Res. Commun., 28, 815 (1967)). When the electrophoretic mobilities were plotted against the logarithm of the known polypeptide chain molecular weights, a smooth curve was obtained. The results show that the method can be used with great confidence to determine the molecular weights of polypeptide chains for a wide variety of proteins.
Excerpt
INTRODUCTION
Following infection of a sensitive bacterium with a phage, a characteristic series of intracellular events occur. In the case of the virulent phage T4, these events include both the cessation of synthesis of many macromolecular constituents characteristic of the growing bacterial cell, and the establishment of a new biosynthetic pattern directed toward the growth and reproduction of the phage. In this new pattern of events, one set of synthetic activities follows another in temporal sequence. For example, a series of enzymes concerned with the synthesis of phage-specific DNA are formed during the first ten minutes following infection while the protein components of the phage particles are synthesized later (see, for example, Kellenberger, 1961). These events are due to the introduction of the phage genome into the bacterial cell and it becomes, therefore, of basic interest to understand how the phage genome is implicated in these processes. This problem which...
Two beginning sequences, pppGpGpGpGpApApCp ... and pppGpGpGpGpGpApApCp ..., have been identified in the RNA of bacteriophage Qbeta. Both were consistently found in nearly equal amounts
The formation of bacteriophage T4 has been studied by characterizing the phage components accumulating in cells infected—under restrictive conditions—with mutants blocked at different stages of the assembly process. Three structures which appear to be intermediates in tail assembly have been isolated by centrifugation: baseplates (S20,w ≅ 80 s), core-baseplates (S20,w ≅ 80 s), and core-baseplates with surrounding sheath (S20,w ≅ 130 s). The functions of genes 19, 48 and 54 are required for the conversion of the baseplate to the core-baseplate. The functions of genes 3, 15 and 18 are required for the formation of the sheath. Gene 18 codes for a major structural protein of the sheath. Genes 3 and 15 specify products required for the stabilization and completion of the sheath.Tail assembly is sequenced; the baseplate is completed first and the core forms on the baseplate. The gene 18 product polymerizes on the core-baseplate and then the 3 and 15 gene products fix the sheath subunits in the polymerized form. After sheath formation, a segment appears to be added to the core at the terminus of the sheath, permitting subsequent attachment of the head. The tail fibers attach only after the particle formed by head-tail union has been acted upon by the gene 9 product. Particles which have not been acted upon by the gene 11 or 12 product adsorb to bacteria but do not kill them.Electron microscopic observations on the state of phage heads in mutant lysates are also presented. Mutations in three genes result in the accumulation of head membranes empty of DNA.Phage heads and phage tails are formed independently of each other.
S ummary
The technique of disc electrophoresis has been presented, including a discussion of the technical variables with special reference to the separation of protein fractions of normal human serum.
Conditional lethal mutations in gene 20 lead to the production of polyheads. Observed intracellularly in sections, polyheads are tubular structures. The cross sections are frequently pentagonal. Polyheads are filled with an unidentified internal substance which is different—at least in concentration—from the contents of normal phage. In a lysate most polyheads are open, but a few are sealed at one or both ends; most open polyheads appear empty upon examination. Serological studies show that polyheads and normal heads have at least one antigenic site in common. The normal heads have at least one more antigenic site than the polyheads.To produce polyheads, the normal functions of two “head” genes are necessary: (a) gene 23 which is believed to produce the protein subunit of the capsid, and (b) gene 31, the function of which has not yet been identified.A temperature-sensitive mutation L65 in gene 23 produces abnormal phage heads. This abnormality is also observed in polyheads produced by the double mutant tsL65 (in gene 23) -amN50 (in gene 20).
When particles of phage T2 are subjected to osmotic shock and the resulting solution is digested with deoxyribonuclease and centrifuged for 5 hours at 93,000g, the supernatant fraction contains 7% of the acid-insoluble sulfur (or about 7% of the total protein) of the particles. The protein fraction (“internal protein”) separated in this way is virtually free from phage coat protein, contains all the internal antigen of the particles, and small amounts of at least one other protein.In solutions prepared by submitting phage particles to osmotic shock but without enzymatic treatment, the internal protein associates reversibly with the nucleic acid at low salt concentrations. It dissociates almost completely at NaCl concentrations greater than 0.1M, at MgCl2 concentrations greater than 0.01M, or at pH greater than 10.In infected bacteria, protein accumulates that is a precursor of the internal protein of phage particles and can be assayed as such by radiochemical methods. Measured in this way, synthesis of internal protein begins promptly after infection, reaches a maximum rate after about 10 minutes, and falls to a somewhat lower rate thereafter. The rate of synthesis is about equal to the rate of incorporation into phage particles after these start to form. The precursor pools contain about 54 phage-equivalent units of internal protein, as compared to 28 units of phage coat protein and 45 units of nucleic acid.Synthesis of internal protein is stopped by addition of chloramphenicol to the culture under conditions that permit continued synthesis of nucleic acid. The rate of incorporation of labeled, early-synthesized internal protein into phage particles is independent of the amount of phage-precursor nucleic acid in the cells when the latter is varied by appropriate chloramphenicol treatment. This is true even when the amount of precursor nucleic acid exceeds the equivalent amount of precursor internal protein, and it shows that if internal protein makes any persistent attachment to phage nucleic acid inside bacteria, it does so at the time of maturation of phage particles, not at the time of synthesis of nucleic acid.Labeled internal protein of the infecting phage particles does not reappear as internal protein in the offspring phage particles.
The protein product of the A cistron (the maturation protein) of bacteriophage R17 has been identified as a structural component of the virus particle. Examination of wild-type R17 labeled with histidine (an amino acid not present in the coat protein of the phage) reveals the existence of one major polypeptide which co-electrophoreses with the A cistron product extracted from actimomycin-treated infected spheroplasts. The A protein, moreover, is totally absent from the uninfective defective particles produced by the growth of three A cistron amber mutants in non-permissive hosts. Gel filtration on Sephadex G200 yields a molecular weight estimate of 35,000 to 40,000 for the histidine-labeled A protein monomer. It can be present in no more than several copies, since on the average only four molecules of histidine are associated with each wild-type phage particle.
A high-frequency transducing element for erythromycin resistance in Staphylococcus aureus has been found. This element, P11de, is apparently the result of a recombinational event between a temperate phage, P11, and a penicillinase plasmid, γ; it lacks substantial sections both of the plasmid and of the bacteriophage. The phage moiety is cryptic, conferring neither lysogeny nor superinfection immunity upon host cells carrying it; helper phage is required for the production of transducing particles but not for the transduction process. Demonstrable phage-related properties include reactivation of ultraviolet-inactivated homologous phage and rescue of temperature-sensitive phage mutants.The plasmic moiety includes an erythromycin resistance marker, and the mc region, which is responsible for plasmid compatibility, is involved in a specific host-plasmid interaction and is required for plasmid replication; all the other known plasmid markers are missing. The autonomous replication of P11de as well as its intracellular behavior vis-à-vis prophages and other plasmids appear to be governed by its mc determinant. In these respects P11de is similar to the parental γ plasmid. This plasmid control, however, is superseded by active P11, which induces P11de to multiply extensively.P11de can recombine with wild-type plasmids; the resulting recombinants are not transduced at high frequency, nor do they reactivate UV inactivated phage particles. Based on these findings, a possible genetic structure for P11de is presented and compared with maps of naturally occurring plasmids.
Enzymatic and genetic evidence are presented for a new pathway of ammonia assimilation in nitrogen fixing bacteria: ammonium → glutamine → glutamate. This route to the important glutamate-glutamine family of amino acids differs from the conventional pathway, ammonium → glutamate → glutamine, in several respects. Glutamate synthetase [(glutamine amide-2-oxoglutarate aminotransferase) (oxidoreductase)], which is clearly distinct from glutamate dehydrogenase, catalyzes the reduced pyridine nucleotide dependent amination of α-ketoglutarate with glutamine as amino donor yielding two molecules of glutamate as product. The enzyme is completely inhibited by the glutamine analogue DON, whereas glutamate dehydrogenase is not affected by this inhibitor; the glutamate synthetase reaction is irreversible. Glutamate synthetase is widely distributed in bacteria; the pyridine nucleotide coenzyme specificity of the enzyme varies in many of these species.
The activities of key enzymes are modulated by environmental nitrogenous sources; for example, extracts of N2-grown cells of Klebsiella pneumoniae form glutamate almost exclusively by this new route and contain only trace amounts of glutamate dehydrogenase activity whereas NH3-grown cells possess both pathways. Also, the biosynthetically active form of glutamine synthetase with a low K
m
for ammonium predominates in the N2-grown cell.
Several mutant strains of K. pneumoniae have been isolated which fail to fix nitrogen or to grow in an ammonium limited environment. Extracts of these strains prepared from cells grown on higher levels of ammonium have low levels of glutamate synthetase activity and contain the biosynthetically inactive species of glutamine synthetase along with high levels of glutamate dehydrogenase. These mutants missing the new assimilatory pathway have serious defects in their metabolism of many inorganic and organic nitrogen sources; utilization of at least 20 different compounds is effected. We conclude that the new ammonia assimilatory route plays an important role in nitrogenous metabolism and is essential for nitrogen fixation.
Studies were performed with tryptophan-operon polarity mutants to determine whether normal numbers of tryp-mRNA molecules were produced by these strains and whether the short tryp-mRNA molecules detected (Imamoto & Yanofsky, 1967) resulted from selective degradation of regions of the intact tryp-mRNA. In experiments in which pulse-labeling was performed immediately after the shift of bacterial cultures from repression to derepression conditions, normal production of early tryp-mRNA was observed in all polarity mutants examined. When pulse-labeling was performed at different times prior to the appearance of the first intact tryp-mRNA molecules in the wild-type strain (before six minutes after the initiation of derepression), reduced levels of tryp-mRNA were detected in the polar mutants and the mRNA region corresponding to the operon region beyond the nonsense codon was conspicuously absent. Since the growing mRNA chain is presumably attached at the site of synthesis during this period, it seems unlikely that selective degradation of intact tryp-mRNA molecules could be responsible for the short mRNA molecules that are detected. It was found that decreasing the pulse period in transcription experiments with polarity mutants failed to increase the relative amount of total detectable tryp-mRNA or the relative level of the tryp-mRNA regions corresponding to the genes of the operon beyond the gene with the polarity mutation. In addition, sucrose gradient sedimentation studies failed to detect a shift of the tryp-mRNA profile of polarity mutants to higher molecular weight regions as the pulse time was decreased. These findings suggest that polarity mutations may cause premature termination of transcription of an operon in the vicinity of an introduced nonsense codon.
Proteins made by E. coli cells infected with bacteriophage T4 were analyzed by a method which combined disc electrophoresis and autoradiography. The precursorproduct relationship between subunits and larger components (head, tail) was studied by taking advantage of the fact that the larger components cannot penetrate into the gel used in disc electrophoresis.These studies have shown that the early proteins are not controlled as a single homogeneous class all members of which are synthesized during the same time periods; instead, they start being formed and are shut off at various times during the early part of the infection process.Amber mutants in gene 30 (polynucleotide ligase-defective) synthesized a small amount of DNA, which was later degraded to a fraction soluble in trichloroacetic acid. The ligase-defective mutants were capable of synthesizing an almost normal amount of late proteins, whereas all other DNA-negative mutants, including a deoxycytidine triphosphatase(dCTPase)-defective mutant and maturation-defective mutants could not induce late protein synthesis. The dCTPase-defective mutant, which also synthesizes a small amount of unstable DNA, did not induce late protein synthesis even when the degradation of DNA was prevented.
By applying analytical acrylamide electrophoresis to degraded purified capsid-related material, we found that (1) normal T4 capsids contain a major component, identified as a product of gene 23, plus at least two minor components, k and l, which we could not yet identify genetically; (2) capsids of the short-headed variant contain the same components as the normal ones; and (3) polyheads contain mainly the product of gene 23 and very little, if any, of the minor components k and l.The minor components can be extracted from capsids by treatment with 8 M urea at 45°. A residual capsid is left behind which, in the electron microscope, is not significantly different from the normal one.It is discussed why k and l are not likely to have a morphopoietic role, since they are identifiable neither with the product of genes 66 and 20, whose morphopoietic functions are known, nor with that of other genes known to be necessary for the production of stable heads.
The subunits of T2 head protein prepared by alkaline or acetic acid degradation form four bands in acrylamide gel electrophoresis. The major band M was confirmed as the product of gene 23. The significance of the k, l, and g bands is considered. T2 and T4 head protein preparations differ in the k band, but not in the M band. The method was not sufficiently sensitive to distinguish the head protein of T4 from that of the amber mutant in gene 23 grown in the permissive cell CR63; nor did it distinguish T2 head protein from the various ht mutants tested. The preparation of stable subunits is discussed.
Capsids of bacteriophage T4 have been dissociated in three different solvents: (1) 6 M guanidine hydrochloride, (2) 6 M guanidine hydrochloride plus 0.1 M β-mercaptoethanol, and (3) 67% acetic acid. Sedimentation equilibrium experiments done in the presence of mercaptoethanol have shown that the capsid subunits can be divided into two classes with molecular weights of approximately 11,000 and 46,000. In the absence of mercaptoethanol, a component of molecular weight 78,000 has been found, indicating that disulfide bonds may be important in stabilizing the phage head structure.
The effect of increasing concentrations of urea on sedimentation velocity, enzymatic activity, antigenicity, and protein fluorescence of ß-galactosidase (ß-d-galactoside galactohydrolase, EC 3.2.1.23) indicates that the enzymatically active tetramer is completely dissociated into inactive monomer in 6 M urea. Removal of urea by dialysis results in reaggregation to the active tetramer together with a return to normal values of those properties which have been studied. Protein which is immunologically related to ß-galactosidase but incapable of forming an enzymatically active tetramer was added to wild-type enzyme in the presence of 8 M urea. After renaturation, the enzymatically active tetramer was found to be a hybrid consisting of mutant and wild-type subunits containing full enzymatic activity.
The complex structure of bacteriophage T4 includes a variety of proteins which become assembled into mature particles during intracellular development of the virus. Some insight into the genetic control of this process has been provided by physiological studies with conditional lethal mutants, which show that over 40 phage genes are involved in T4 morphogenesis (Fig. 1). However, the mechanisms by which components are assembled have remained obscure, due in part to the lack of a suitable system for their study. In the experiments reported below, conditional
lethal mutants of strain T4D have been exploited to develop an in vitro system in which several of the steps in phage morphogenesis can be demonstrated.
Ornstein and Davis (1964) have introduced a method, called “disc electrophoresis”, which yields extremely high resolution of proteins electrophoresed in cylindrical columns of polyacrylamide gel. By means of disc electrophoresis, a large number of protein components in a complex mixture can be separated and detected in a single operation. Bands of C14-labeled proteins in disc electropherograms can be detected using techniques described by Heideman (1964); and Jovin, Chrambach, and Naughton (1964). This report presents an alternative procedure involving autoradiography of dried longitudinal gel slices. The method is relatively uncomplicated and can be used to develop the entire pattern of radioactivity in a gel without significant sacrifice of resolution.
Three acid-soluble components have been detected in E. coli infected with bacteriophage T4D. These components first appear in infected cells at 11 minutes after infection (at 37 °), and two of them are incorporated into mature phage particles from which they can be released by osmotic shock as well as by trichloroacetic acid extraction. The properties of these components indicate that they are polypeptides of several thousand molecular weight. They thus appear to correspond, at least in part, to the acid-soluble peptide fraction in T2H phage particles described earlier by Hershey (1957).Conditionally lethal (amber) mutants of T4D blocked in head formation fail to produce all three of the components in a nonpermissive host. This is true for mutants affected in any of six different genes. By contrast, mutants blocked in other kinds of assembly functions are able to produce the components. This finding suggests that the appearance of the components is associated with formation of the phage head.A delay of several minutes between the incorporation of a labeled amino acid into protein and the appearance of label in the acid-soluble components indicates that that these components arise from a precursor.These findings suggest that during phage maturation a protein is encapsulated with the phage DNA and is subsequently fragmented to yield the acid-soluble components which remain trapped inside the phage head. Some possibilities are discussed concerning the relation of this process to phage maturation.
The physiological manifestation of conditional lethal mutations in the genes involved in the formation of the T4 head have been re-examined. If the product of genes 20 or 40 is defective, open-ended tubular structures (polyheads) are synthesized. Infection with mutants in gene 22 leads to the formation of polyheads, about 70% of which consist of two or more concentric layers (multilayered polyheads). Cells infected with mutants in gene 21 and 24 yield a mixed burst of capsid-like particles (τ-particles) and polyheads, many of which have hemispherical caps.
Two structures related to the head of bacteriophage T4, polyheads and τ-particles, are shown by electron microscopy to contain an internal component defined as a core. These cores can show a high degree of organization.Multilayered polyheads are described which contain a normal sized core.The dark polyhedral bodies seen in sections and previously referred to as DNA “condensates” were reinvestigated, and were found to have a visible membrane. However, this membrane is very fragile and does not survive lysis of the cell in a recognizable form.The appearance of thin sections of T4 heads obtained after different fixation methods suggests that the “less dense areas,” which can be observed when fixed under conditions found to be inadequate for DNA fixation, are likely to be artifacts and therefore cannot be used alone as proof for the presence of a core.We conclude that the present information from morphological, genetic, and biochemical experiments strongly suggests that the observed cores have a morphopoietic role. We discuss the structure and possible mechanism of action of such a core.
In reply to recent criticism Professor Commoner discusses current evidence in support of his conclusion that the Watson-Crick theory is an inadequate explanation of inheritance.
The kinetics of the assembly of polyheads produced by infecting Escherichia coli B with T4amber mutants in gene 20 was measured and compared with the growth of wild type phage. The rates of production of polyheads and of phages were found to be about the same. The final yields in lysis-inhibited cells were approximately 600 phage equivalents per infected bacterium. The initial appearance of polyheads is delayed 15–20 min compared with wild type phage production, although it is not due to a reduced rate of protein synthesis in mutant-infected cells. In such cells an accumulation of precursor protein for polyhead is thus caused. This pool is about three times larger than the one measured during wild type infection. The delay is extended if the amount of subunits available for polyhead formation is reduced. We conclude that the initiation of polyhead assembly depends upon the subunit concentration. Polyhead assembly continues at the same rate for several minutes when protein synthesis is inhibited with chloramphenicol at different times. The maturable polyhead precursor was estimated by measuring the amount of polyheads assembled after adding the drug, and it was found that 25% of the total protein pool was converted into polyheads. Using a new technique for the observation of single cells with the electron microscope we found that polyheads are arranged in bundles oriented parallel to the long axis of the cell. The average length of polyheads is roughly the same at all times during their formation.
Fundamental Techniques of Virology
- J. V. Maizel
Fundamental Techniques of Virology (edit
- J V Maizel
- JV Maizel