Background β-1,3-Glucanases catalyze the hydrolysis of glucan polymers containing β-1,3-linkages. These enzymes are of great biotechnological, agricultural and industrial interest. The applications of β-1,3-glucanases is well established... more
Background β-1,3-Glucanases catalyze the hydrolysis of glucan polymers containing β-1,3-linkages. These enzymes are of great biotechnological, agricultural and industrial interest. The applications of β-1,3-glucanases is well established in fungal disease biocontrol, yeast extract production and wine extract clarification. Thus, the identification and characterization of novel β-1,3-glucanases with high catalytic efficiency and stability is of particular interest. Results A β-1,3-glucanase gene designated PglA was cloned from a newly isolated strain Paenibacillus sp. S09. The gene PglA contained a 2631-bp open reading frame encoding a polypeptide of 876 amino acids which shows 76% identity with the β-1,3-glucanase (BglH) from Bacillus circulans IAM1165. The encoded protein PglA is composed of a signal peptide, an N-terminal leader region, a glycoside hydrolase family 16 (GH16) catalytic domain and a C-terminal immunoglobulin like (Ig-like) domain. The Escherichia coli expression sys...
Research Interests: Biochemistry, Biology, Enzyme Kinetics, Biological Sciences, Environmental Sciences, and 15 moreCell fusion, Escherichia coli, Animals, Alkaline phosphatase, Enzyme, Amino Acids, Antibody, Biologicals, Acinetobacter baumannii, Amino Acid Sequence, Antineoplastic Agents, Extracellular, Biochemistry and cell biology, Bioresource technology, and Cricetinae
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Highly specific protein–protein interactions feature in all areas of biochemistry. The genome sequences now available are increasing both our knowledge of the protein composition of cells and the need to understand how they interact.... more
Highly specific protein–protein interactions feature in all areas of biochemistry. The genome sequences now available are increasing both our knowledge of the protein composition of cells and the need to understand how they interact. Keywords: binding; affinity; coiled-coils; antibody; quaternary structure
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Cancer is one of the most prevalent diseases in the world. Breast cancer is the second most deadly cancer type after lung cancer. Surgical intervention, chemotherapy and radiotherapy are the most used conventional methods in the treatment... more
Cancer is one of the most prevalent diseases in the world. Breast cancer is the second most deadly cancer type after lung cancer. Surgical intervention, chemotherapy and radiotherapy are the most used conventional methods in the treatment of breast cancer. The non-targeted approach of conventional treatments causes serious side effects in healthy cells and tissues, and often mortality is due to the side effects of these conventional treatments. In recent years, nano-sized particles called drug delivery systems targeting cancer cells have attracted attention as a new approach in cancer treatment. The fact that these nanocarrier systems target tumor cells without damaging healthy tissues has been a hope for breast cancer. Moreover, nanocarriers are unique biomaterials that may exhibit low toxicity, high biocompatibility, biodegradability, ease of use, high dose drug loading, and adjustable surface functionalities. In the present study, we summarize recent studies of nanocarriers that offer a critical review of an alternative strategy to breast cancer therapy.
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Heat shock protein 90 (Hsp90) is a key chaperone that is abnormally expressed in cancer cells, and therefore, designing novel compounds to inhibit chaperone activities of the Hsp90 is a promising therapeutic approach for cancer drug... more
Heat shock protein 90 (Hsp90) is a key chaperone that is abnormally expressed in cancer cells, and therefore, designing novel compounds to inhibit chaperone activities of the Hsp90 is a promising therapeutic approach for cancer drug discovery. Debio-0932 is a second-generation Hsp90 inhibitor that exhibited promising anticancer activity against a wide variety of cancer types with a strong binding affinity for Hsp90 and high oral bioavailability. Anticancer activities of the Debio-0932 were tested in MCF-7 and MDA-MB-231 cell lines. Molecular docking results indicated that Debio-0932 was selectively bound to the ATP binding pocket of the Hsp90 with an estimated free energy of binding - 7.24 kcal/mol. Antiproliferative activity of Debio-0932 was determined by XTT assay and Debio-0932 exhibited a cytotoxic effect on MCF-7 and MDA-MB-231 cells in a time and dose-depended manner. Apoptosis inducer role of Debio-0932 was evaluated in MCF-7 and MDA-MB-231 cells with fluorometric apoptosis/necrosis detection kit. Treatment with Debio-0932 stimulated apoptosis in both breast cancer cell lines. mRNA and protein expression levels of Bax, Bcl-2 and Casp-9 were determined in MCF-7 and MDA-MB-231 cells by RT-PCR and Western blotting respectively. Debio-0932 stimulated the down-regulation of anti-apoptotic protein Bcl-2 and the up-regulation of apoptotic protein Bax and cleavage of Casp-9 in cancer cells. Moreover, the anti-invasive potential of Debio-0932 was evaluated in endothelial cells (HUVEC) by wound-healing assay. Debio-0932 decreased the migration of HUVEC cells as compared to the control group. These results indicate that Debio-0932 is a promising compound to treat triple-negative breast cancer and hormone receptor-positive breast cancer, and their metastases.
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Abstract The green synthesis of metal nanoparticles has been promising approach in the field of nanomedicine due to its low-cost, non-toxic, biocompatible and sustainable features. In literature, therapeutic potentials of silver... more
Abstract The green synthesis of metal nanoparticles has been promising approach in the field of nanomedicine due to its low-cost, non-toxic, biocompatible and sustainable features. In literature, therapeutic potentials of silver nanoparticles (AgNPs) coupled with natural extracts have been extensively evaluated against various diseases. The present study aims at preparation of AgNPs using Coriolus versicolor and Boletus edulis mushroom extract based on the microwave-assisted method. The synthesized C. versicolor-silver nanoparticles (CV-AgNPs) and B. edulis-silver nanoparticles (BE-AgNPs) were characterized by UV–Visible spectroscopy, dynamic light scattering technique (DLS) and fourier transform infrared spectroscopy (FTIR). Anticancer activity of AgNPs was tested on MCF-7, HT-29 and HUH-7 cell lines. Antimicrobial efficacy of AgNPs was determined against pathogens bacterial (Pseudomonas aeruginosa, Klebsiella pneumonia, Staphylococcus aureus and Enterococcus faecalis) and fungal (Candida albicans and Candida utilis) strains. Also, wound healing potential of AgNPs was examined on L929 cells. Obtained results demonstrated that synthesized CV-AgNPs and BE-AgNPs had an average size diameter of
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Endoglucanases randomly hydrolyse the cellulose chains by acting upon internal β-1,4-D-glycosidic bonds and are used extensively in industrial applications. In this study, bacterial endoglucanase gene yhfE was obtained by PCR, using... more
Endoglucanases randomly hydrolyse the cellulose chains by acting upon internal β-1,4-D-glycosidic bonds and are used extensively in industrial applications. In this study, bacterial endoglucanase gene yhfE was obtained by PCR, using primers based on genomic sequences of Bacillus subtilis strains. 1041 bp DNA fragment of yhfE was cloned into Escherichia coli DH5α through the use of pTolT expression plasmid. PCR, restriction enzyme analysis and DNA sequencing were performed in order to confirm the cloning. E. coli BL21-AI cells expressed the yhfE after induction at 0.04% of arabinose concentration for 4 h. The expected 38.7 kDa size yhfE protein after digestion with thrombin of the His-tagged fusion protein (yhfE-TolAIII) was visualized by SDS-PAGE. The yhfE-TolAIII production yield was approximately 82 mg/L. The recombinant yhfE was characterized by MALDI-TOF mass spectrometry and CD analysis.
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In recent years, various studies in the field of industrial enzymes of biotechnology have gained importance due to increasing development in enzyme technology. The variety of areas were enzymes are used and economic value of... more
In recent years, various studies in the field of industrial enzymes of biotechnology have gained importance due to increasing development in enzyme technology. The variety of areas were enzymes are used and economic value of biotechnological products further increase their importance. There are hundreds of different types of cheese but each is made by coagulating milk using rennet to give curds. Today, researchers have begun to develop alternative systems in the cheese industry as related to milk clotting enzymes. In this study the nucleic acid sequence encoding the optimized chymosin enzyme was used and cloned by Not I and Mlu I restriction enzymes into pTOLT vector system. Then using this construct the enzyme as a fusion with Tol-A-III protein was produced in Escherichia coli BL21 (DE3) cells. After disrupting the E. coli cell and separating from the constituents by high speed centrifugation the enzyme was purified by affinity chromatography and fractions were analysed by SDS-PAGE. Purified enzyme has shown activity. Optimum temperature and pH of CHY-Tol-A-III protein were 40 °C and 6.5, respectively.
Research Interests: Biochemistry, Technology, Biology, Medicine, Biological Sciences, and 14 moreEscherichia coli, Animals, Temperature, Plasmids, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Affinity chromatography, Enzyme, CHEMICAL SCIENCES, Molecular cloning, Base Sequence, Rennet, Hydrogen-Ion Concentration, proteolysis, and Chymosin
Over the last decade, microalgae has drawn attention as a natural source of valuable compounds and as bioreactors for recombinant protein production. Microalgae-based bioreactor is newly employed for production of safe and cost effective... more
Over the last decade, microalgae has drawn attention as a natural source of valuable compounds and as bioreactors for recombinant protein production. Microalgae-based bioreactor is newly employed for production of safe and cost effective proteins. Especially, Chlamydomonas reinhardtii , an unicellular microalga, is the most prominent species which has a short generation time, fast growth rate, multiple genetic systems, and ability to perform posttranslational modifications machinery that plays a significant role in regulating the activity of complex proteins. In this research, the nuclear genome of the Chlamydomonas reinhardtii CC-125 strain was transformed by electroporation using construct plasmid pChlamy_3-GFP containing the gene coding for Green Fluorescent Protein (GFP) which is commonly used as an universal marker in biotechnological studies. Molecular and genetic analyses conducted on transformants revealed that the nuclear genome was stably transformed and the transgenes were integrated into the algal chromosomal DNA succesfully, albeit there was no distinct expression level of GFP gene in producing large amounts of protein. Codon optimization, choice of promoters, introns and UTRs, endogenous enhancer elements, regulatory mechanisms, localization of proteins, posttranslational modifications and protease activities are the possible underlying causes of the low expression level. Key words: Chlamydomonas reinhardtii, bioreactor, green fluorescent protein, electroporation Model Organizma Chlamydomonas reinhardtii 'ye Elektroporasyon Araciligiyla GFP Geninin Transferi OZET : Son on yilda, mikroalgler degerli bilesiklerin dogal kaynagi ve rekombinant protein uretimi icin biyoreaktor olmalari sebebiyle dikkat cekmektedirler. Guvenli ve ucuz protein uretimi icin, mikroalg tabanli biyoreaktorler yeni yeni kullanilmaktadir. Ozellikle de kisa ureme suresine, hizli buyume oranina, coklu genetik sisteme ve kompleks proteinlerin aktivite kazanmalari icin gereken post translasyonel modifikasyon mekanizmalarina sahip tek hucreli mikroalg turu Chlamydomonas reinhardtii one cikmaktadir. Bu calismada, biyoteknolojik calismalarda evrensel markor olarak kullanilan GFP genini iceren konstrukt pChlamy_3-GFP plazmidi, Chlamydomonas reinhardtii CC-125 susunun nuklear genomuna elektroporasyon yontemi ile aktarilmistir. Transformantlarin molekuler ve genetik analizleri, GFP geninin yuksek miktarda belirgin bir ekspresyon seviyesi olmamasina ragmen nuklear genomun stabil olarak transforme edildigini, transgenlerin, alg kromozomal DNA'sina basarili bir sekilde entegre oldugunu gostermistir. Kodon optimizasyonu, promotor secimi, intron ve UTR'ler, endojen enhansir elemanlari, duzenleyici mekanizmalar, proteinlerin lokalizasyonu, posttranslasyonel modifikasyonlar ve proteaz aktiviteleri, dusuk ekspresyon seviyesinin altinda yatan muhtemel nedenlerdir. Anahtar kelimeler : Chlamydomonas reinhardtii, biyoreaktor, yesil floresan protein, elektroporasyon
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The increase in the frequency of drug resistance in bacterial infections has led to the research of new antibacterial agents targeting new mechanisms. Many of the functions of NAD+-dependent DNA ligase have made it a remarkable target for... more
The increase in the frequency of drug resistance in bacterial infections has led to the research of new antibacterial agents targeting new mechanisms. Many of the functions of NAD+-dependent DNA ligase have made it a remarkable target for antibacterial drug discovery. Escherichia coli (E. coli) NAD+-dependent DNA ligase is presented as a potential target due to its unique substrate specificity compared to the ATP-dependent human DNA ligase. In this study, it was aimed to produce and purify the E. coli NAD + dependent DNA ligase enzyme, which is frequently used in antibacterial drug discovery. The E. coli DNA ligase gene sequence was cloned into pTOLT vector system. E. coli DNA ligase enzyme was purified after the production in E. coli BL21 (DE3) pLysE cells. It was clearly demonstrated by the activity test that the DNA ligase enzyme produced in this study can ligate the DNA fragments. As a result, it was revealed that the effect of candidate inhibitors can be studied simply on the e...
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Genome editing is a method used to make desired changes in the target gene. Today, various methods are used for genome-editing studies; among them, one of the most widely used methods is the clustered, regularly interspaced short... more
Genome editing is a method used to make desired changes in the target gene. Today, various methods are used for genome-editing studies; among them, one of the most widely used methods is the clustered, regularly interspaced short palindromic repeats (CRISPR). CRISPR-associated (Cas) genes and their corresponding CRISPR sequences constitute CRISPR-Cas systems. Due to its simplicity, it is likely that the CRISPR–Cas system could be used effectively in ex vivo gene therapy studies in humans. If this happens, the importance of CRISPR carrier systems will gradually increase. Viral and non-viral systems are used as delivery modalities in genome-editing studies. It has been proven that nanoparticles are the most promising tools for gene therapy due to their adjustable size, surface, shape, and biological behaviours. The polymeric carrier system has become the main non-viral substitute for gene delivery due to its reduced immunogenicity and pathogenicity. In this review, information about c...
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Vascular endothelial growth factor 165 (VEGF165) is the best characterized member and most potent endothelial cell mitogenic factor belonging to the VEGF family. Because VEGF165 is the major player in angiogenesis in vivo and has... more
Vascular endothelial growth factor 165 (VEGF165) is the best characterized member and most potent endothelial cell mitogenic factor belonging to the VEGF family. Because VEGF165 is the major player in angiogenesis in vivo and has potential in therapeutic applications for accelerating wound repair, many expression systems have been developed to produce VEGF165. In this study, biological active recombinant human VEGF165 (rhVEGF165) was expressed for the first time in Kluyveromyces lactis GG799 cells. The gene encoding human VEGF165 was cloned in K. lactis genome by homologous recombination. The VEGF165 was secreted in culture medium at a level of 5.7 mg/L and expression was confirmed by SDS-PAGE and Western blotting. Downstream purification was performed using ammonium sulphate precipitation and affinity chromatography. The biological activity of rhVEGF165 was tested by the proliferation assay on the human umbilical vein-derived endothelial cells (HUVEC) in a dose and time-dependent manner. The K. lactis-derived rhVEGF165 exhibited proliferative activity with a half-maximal effective concentration of 3.0. Cell migration analysis was conducted to evaluate the in vitro wound healing effect of the produced rhVEGF165 via a scratch assay. These findings indicate that K. lactis could be a suitable host for secreting bioactive human VEGF165 for therapeutic use.
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Colicins are bacterial toxins that kill Escherichia coli and related cells; their mode of action is of interest in protein import and toxicology. Colicins translocate across the E. coli outer membrane and periplasm by interacting with... more
Colicins are bacterial toxins that kill Escherichia coli and related cells; their mode of action is of interest in protein import and toxicology. Colicins translocate across the E. coli outer membrane and periplasm by interacting with several receptors. This translocation process involves interaction of the colicin with the outer membrane porin OmpF and subsequently with the integral membrane protein TolA. The N-terminal domain of colicin N is involved in the import process. Our aim was to produce a large quantity of colicin A for functional and structural studies. It is a prerequisite to have a correctly folded and stable protein for these studies. The commonly utilised expression system uses the Lex A promoter, which requires induction with toxic mitomycin C, though the yield is low. Here we present the production of an E. coli toxin and its immunity protein in E. coli using a safe inducible promoter.
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Chymosin is a very important industrial enzyme that commonly used in cheese manufacture. Bovine chymosin is an aspartic protease which is extracted from abomasum of suckling calves. In this study, bovine chymosin gene was first optimized... more
Chymosin is a very important industrial enzyme that commonly used in cheese manufacture. Bovine chymosin is an aspartic protease which is extracted from abomasum of suckling calves. In this study, bovine chymosin gene was first optimized and then cloned into pTOLT E.coli expression system for production of chymosin. Protein engineering of chymosin has also been attempted. A number of companies are now producing recombinant chymosin for commercial use in cheese manufacture
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Over the last decade, microalgae has drawn attention as a natural source of valuable compounds and as bioreactors for recombinant protein production. Microalgae-based bioreactor is newly employed for production of safe and cost effective... more
Over the last decade, microalgae has drawn attention as a natural source of valuable compounds and as bioreactors for recombinant protein production. Microalgae-based bioreactor is newly employed for production of safe and cost effective proteins. Especially, Chlamydomonas reinhardtii , an unicellular microalga, is the most prominent species which has a short generation time, fast growth rate, multiple genetic systems, and ability to perform posttranslational modifications machinery that plays a significant role in regulating the activity of complex proteins. In this research, the nuclear genome of the Chlamydomonas reinhardtii CC-125 strain was transformed by electroporation using construct plasmid pChlamy_3-GFP containing the gene coding for Green Fluorescent Protein (GFP) which is commonly used as an universal marker in biotechnological studies. Molecular and genetic analyses conducted on transformants revealed that the nuclear genome was stably transformed and the transgenes wer...
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New and improved genetic engineered variants of fluorescent proteins (FPs) have become useful tools for bioimaging in biomedical researches. Red fluorescent proteins (RFPs) first derived from the sea anemone Discosoma show high... more
New and improved genetic engineered variants of fluorescent proteins (FPs) have become useful tools for bioimaging in biomedical researches. Red fluorescent proteins (RFPs) first derived from the sea anemone Discosoma show high performance in vivo labeling and imaging. mCherry is a member of RFPs which has very high photostability, resistant to photo bleaching and rapid maturation. These advantages ensure that mCherry can be successfully fused to many proteins and widely used for quantitative imaging techniques. In this study, the constructed recombinant plasmid pBADCherry was expressed in Escherichia coli BL21(AI) then culture conditions, inducer concentration and induction time were optimized. Results of sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS - PAGE ) analysis demonstrated that 5 hours induction at 0.04% of arabinose concentration was optimal for the highest mCherry yield. The expression of hexa histidine-tagged (6xHis) recombinant mCherry was induced by a...
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Bu calismada; proteinlerin elektroforez jellerinin boyanmasinda dogal boyalarin kullanilabilirligi arastirilmistir. Bu amacla; ceviz (Juglans regia), karadut (Morus nigra), kokboya (Rubia tinctorum), karamuk (Berberidis crataegina) ve... more
Bu calismada; proteinlerin elektroforez jellerinin boyanmasinda dogal boyalarin kullanilabilirligi arastirilmistir. Bu amacla; ceviz (Juglans regia), karadut (Morus nigra), kokboya (Rubia tinctorum), karamuk (Berberidis crataegina) ve kizilagac (Alnus glutonisa) kullanilmistir. Bu bitkilerden hazirlanan materyal ile kan proteinlerinin kosuldugu SDS PAGE jelleri boyanmistir. 9x6 cm buyuklugundeki jellere kan proteinlerinden; albumin, globulin, hemoglobin, fibrinojen kosulmus, ardindan bu jellere dogal boyalar uygulanmis ve Coomassie Brillant Blue ile boyanmis olan kontrol jeli ile karsilastirilmistir. Sonuc olarak; protein bantlarinin gozlenmesi icin elektroforez jellerinin boyanmasinda dogal boyalardan bazilari kullanilabilir
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L'invention concerne des proteines de fusion (polypeptides de fusion), particulierement utiles dans des systemes d'expression et/ou de purification. Les inventeurs de la presente invention ont trouve que le domaine de TolAIII... more
L'invention concerne des proteines de fusion (polypeptides de fusion), particulierement utiles dans des systemes d'expression et/ou de purification. Les inventeurs de la presente invention ont trouve que le domaine de TolAIII presentait d'excellentes proprietes particulierement appropriees a une utilisation en tant que partenaire de proteine de fusion afin d'obtenir des niveaux eleves d'expression dans une cellule hote. Dans un aspect de l'invention, un domaine de TolAIII ou un homologue, fragment, ou derive fonctionnel dudit domaine est situe a proximite du N-terminal dudit polypeptide de fusion et un polypeptide non-TolA est situe a proximite du C-terminal dudit polypeptide de fusion.
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Abstract The dramatic increase in the atmospheric carbon dioxide levels and the apprehensive situation of energy supply are leading to the importance of renewable energy technology studies. Because solar energy is a sustainable energy... more
Abstract The dramatic increase in the atmospheric carbon dioxide levels and the apprehensive situation of energy supply are leading to the importance of renewable energy technology studies. Because solar energy is a sustainable energy source, developments in photovoltaic technologies are continuing rapidly. This section describes the biologically improved materials for photovoltaics application technology in detail. Biomaterials and natural compounds contribute to the sustainability of organic electronics because of the advantages of being environmentally friendly and naturally available. Despite these advantages, very few biomaterials have been included in organic-based solar cell production studies. For photovoltaic technologies, improvements or syntheses should be made by considering many physical properties such as hardness, elasticity, elongation coefficient, thermal strength point, optical properties, and long-term strength of candidate biomaterials. Therefore design, development or selection of special biomaterials can only be achieved with the collaboration of scientists in the materials science and bioengineering fields. While polymeric biomaterials used in the photovoltaic technology are generally polypeptide structured, applications with green technology such as microorganisms, pigments, and other biological systems (Photosystem I and Photosystem II) are rapidly growing. In this chapter, the processes of obtaining biomaterials and ways to improve productivity and stability to be used in photovoltaic technology by natural and/or synthetic biology approaches, will be discussed.
Heavy metals are considered to be the most important pollutants in the contamination of soils; they adversely affect plant growth and development and cause some physiological and molecular changes. The contamination of agricultural soils... more
Heavy metals are considered to be the most important pollutants in the contamination of soils; they adversely affect plant growth and development and cause some physiological and molecular changes. The contamination of agricultural soils by heavy metals has changed the mineral element content of vegetables. Plant metallothioneins (MTs) are thought to have the functional role in heavy metal homeostasis, and they are used as the biomarkers for evaluating environmental pollution. We aimed to evaluate the expression of MT isoforms (MT1, 2, 3 and 4) and some mineral element composition of tomato roots, leaves and fruits exposed to copper and lead. Heavy metal applications increased MT1 and MT2 gene expressions compared to the control in the tissues of tomato. The highest level of MT1 and MT2 transcripts was found in roots and leaves, respectively. The expression of MT3 is induced in roots, leaves and fruits except for Pb treatment in roots. MT4 expression increased in fruits; however, other tissues did not show a clear change. Our results indicated that Cu content was higher than Pb in all tissues of tomato. The lower doses of Cu (10 ppm) increased the content of Mg, Fe, Ca and Mn in roots. Pb generally increased the level of minerals in leaves and fruits, but it decreased Mg, Mn and Fe contents in roots. Both heavy metals not only moved to aerial parts but also caused alterations to mineral element levels. These results show that MT transcripts are regulated by Cu and Pb, and expression pattern changes to MT isoforms and tissue types. © 2016 Society of Chemical Industry.
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In recent years, various studies in the field of industrial enzymes of biotechnology have gained importance due to increasing development in enzyme technology. The variety of areas were enzymes are used and economic value of... more
In recent years, various studies in the field of industrial enzymes of biotechnology have gained importance due to increasing development in enzyme technology. The variety of areas were enzymes are used and economic value of biotechnological products further increase their importance. There are hundreds of different types of cheese but each is made by coagulating milk using rennet to give curds. Today, researchers have begun to develop alternative systems in the cheese industry as related to milk clotting enzymes. In this study the nucleic acid sequence encoding the optimized chymosin enzyme was used and cloned by Not I and Mlu I restriction enzymes into pTOLT vector system. Then using this construct the enzyme as a fusion with Tol-A-III protein was produced in Escherichia coli BL21 (DE3) cells. After disrupting the E. coli cell and separating from the constituents by high speed centrifugation the enzyme was purified by affinity chromatography and fractions were analysed by SDS-PAGE...
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Highly specific protein–protein interactions feature in all areas of biochemistry. The genome sequences now available are increasing both our knowledge of the protein composition of cells and the need to understand how they interact.... more
Highly specific protein–protein interactions feature in all areas of biochemistry. The genome sequences now available are increasing both our knowledge of the protein composition of cells and the need to understand how they interact. Keywords: binding; affinity; coiled-coils; antibody; quaternary structure
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A method is presented to produce large amounts of Bcl-2 and Bcl-x(L), two anti-apoptotic proteins of considerable biomedical interest. Expression constructs were prepared in which the Escherichia coli protein TolAIII, known to promote... more
A method is presented to produce large amounts of Bcl-2 and Bcl-x(L), two anti-apoptotic proteins of considerable biomedical interest. Expression constructs were prepared in which the Escherichia coli protein TolAIII, known to promote over expression of soluble product, was added to the N-terminus of Bcl-2 or Bcl-x(L) proteins, which had their C-terminal hydrophobic anchors deleted. Here the expression of these TolAIII-fusion constructs, followed by a two-step metal-affinity based purification protocol is described. The method delivers at least 20 and 10mg of more than 90% pure TolAIII-Bcl-x(L)DeltaC and TolAIII-Bcl-2(2)DeltaC proteins, respectively, per liter of E. coli cell culture. The proteins are released by proteolysis with thrombin providing > 12 mg of Bcl-x(L)DeltaC or > 6 mg of Bcl-2(2)DeltaC per liter of E. coli cell culture with a purity of more than 95%. Whereas Bcl-x(L)DeltaC is soluble both before and after TolAIII removal, Triton X-100 can significantly increase the extraction of TolAIII- Bcl-2(2)DeltaC from the bacterial cells and its subsequent solubility. Far-UV CD spectroscopy demonstrated that they both have an alpha-helical structure. Fluorescence spectroscopy was used to quantitatively analyze the binding of the respiratory inhibitor antimycin A to recombinant Bcl-2 and Bcl-x(L) proteins as well as the displacement of this ligand from the hydrophobic pocket with BH3 Bad-derived peptide. Purified Bcl-x(L)DeltaC and Bcl-2(2)DeltaC both protect isolated mitochondria from Bax-induced release of cytochrome c. The ensemble of data shows that the expressed proteins are correctly folded and functional. Therefore, the TolAIII-fusion system provides a convenient tool for functional characterization and structural studies of anti-apoptotic proteins.
Research Interests: Fluorescence Spectroscopy, Biology, Mitochondria, Apoptosis, Medicine, and 14 moreCell Culture, Circular Dichroism, Escherichia coli, Animals, Nmr, Cytochrome C, Plasmids, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Protein expression and purification, Rats, Wistar Rats, Base Sequence, Fusion Protein, and Biochemistry and cell biology
We have fabricated a molecular organic light-emitting device comprising indium–tin oxide/a molecular organic layer/aluminum in which the organic layer is a green fluorescent protein. The device exhibits peak external quantum and power... more
We have fabricated a molecular organic light-emitting device comprising indium–tin oxide/a molecular organic layer/aluminum in which the organic layer is a green fluorescent protein. The device exhibits peak external quantum and power efficiencies of 8 ± 0.2% and 13 ± 0.7 lm W at a current of J = 1.5 A m, respectively. In addition, the turn-on voltage is 2.5 V for 1 cd m and the maximum luminance achieved is 1275 cd m. This