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cell free protein synthesis

Mohammed I El-Gamal, Woo-Seok Lee, Ji-Sun Shin, Chang-Hyun Oh, Kyung-Tae Lee, Jungseung Choi, Nohsun Myoung, Daejin Baek
The synthesis of a new series of 21 fused coumarin derivatives is described, and the biological evaluation of their in vitro antiinflammatory effects as inhibitors of lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2 ) production in RAW 264.7 macrophages. The target compounds 1a-u were first tested for cytotoxicity to determine a non-toxic concentration for antiinflammatory screening, so that the inhibitory effects against NO and PGE2 production would not be caused by cytotoxicity...
October 24, 2016: Archiv der Pharmazie
Nitzan Krinsky, Maya Kaduri, Janna Shainsky-Roitman, Mor Goldfeder, Eran Ivanir, Itai Benhar, Yuval Shoham, Avi Schroeder
Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa)...
2016: PloS One
Zhu Zeng, Rui Yu, Fanglei Zuo, Bo Zhang, Deju Peng, Huiqin Ma, Shangwu Chen
Exendin-4, a glucagon-like protein-1 (GLP-1) receptor agonist, is an excellent therapeutic peptide drug for type 2 diabetes due to longer lasting biological activity compared to GLP-1. This study explored the feasibility of using probiotic Lactobacillus paracasei as an oral vector for recombinant exendin-4 peptide delivery, an alternative to costly chemical synthesis and inconvenient administration by injection. L. paracasei transformed with a plasmid encoding the exendin-4 gene (L. paracasei L14/pMG76e-exendin-4) with a constitutive promotor was successfully constructed and showed efficient secretion of exendin-4...
2016: PloS One
Da Zhou, Bing-Hang Li, Jing Wang, Yong-Nian Ding, Yan Dong, Yuan-Wen Chen, Jian-Gao Fan
BACKGROUND: Prolyl oligopeptidase (POP) is a serine endopeptidase that is widely distributed in vivo, particularly in the liver. Significant changes in functional mitochondrial proteins involved with mitochondrial oxidoreductases/transporters and nucleic acid binding proteins were observed after POP inhibition in the liver, which suggested a role of POP in regulating liver energy metabolism. Steatosis in nonalcoholic fatty liver disease (NAFLD) is associated with disturbances in lipid and energy metabolism in hepatocytes...
2016: PloS One
Katsuaki Hirano, Masahiro Kurosaki, Satoshi Nihei, Hiroki Hasegawa, Suguru Shinoda, Mitsuru Haruki, Nobutaka Hirano
The cellulosome is a supramolecular multienzyme complex comprised of a wide variety of polysaccharide-degrading enzymes and scaffold proteins. The cellulosomal enzymes that bind to the scaffold proteins synergistically degrade crystalline cellulose. Here, we report in vitro reconstitution of the Clostridium thermocellum cellulosome from 40 cellulosomal components and the full-length scaffoldin protein that binds to nine enzyme molecules. These components were each synthesized using a wheat germ cell-free protein synthesis system and purified...
October 19, 2016: Scientific Reports
Bartosz Gląb, Mirela Beganovic, Sanket Anaokar, Meng-Shu Hao, Allan Rasmusson, Jana Patton-Vogt, Antoni Banaś, Sten Stymne, Ida Lager
Glycero-3-phosphocholine (GPC), the product of the complete deacylation of phosphatidylcholine (PC), was long thought to not be a substrate for reacylation. However it was recently shown that cell-free extracts from yeast and plants could acylate GPC with acyl groups from acyl-CoA. By screening enzyme activities of extracts derived from a yeast knock-out collection, we were able to identify and clone the yeast gene (GPC1) encoding the enzyme, named glycerophosphocholine acyltransferase (GPCAT). By homology search we also identified and cloned GPCAT genes from three plant species...
October 7, 2016: Journal of Biological Chemistry
Siddiq Pasha Shaik, V Lakshma Nayak, Faria Sultana, A V Subba Rao, Anver Basha Shaik, Korrapati Suresh Babu, Ahmed Kamal
A series of imidazo[2,1-b]thiazole linked triazole conjugates were synthesized by using Huisgen 1,3-dipolar cyclo-addition reaction (click chemistry approach) and evaluated for their antiproliferative activity against some human cancer cell lines like, HeLa (cervical), DU-145 (prostate), A549 (lung), MCF-7 (breast) and HepG2 (liver). Among them, Conjugates 4g and 4h demonstrated a significant antiproliferative effect against human lung cancer cells (A549) with IC50 values of 0.92 and 0.78 μM respectively...
September 20, 2016: European Journal of Medicinal Chemistry
Jessica G Perez, Jessica C Stark, Michael C Jewett
Cell-free protein synthesis (CFPS) technologies have enabled inexpensive and rapid recombinant protein expression. Numerous highly active CFPS platforms are now available and have recently been used for synthetic biology applications. In this review, we focus on the ability of CFPS to expand our understanding of biological systems and its applications in the synthetic biology field. First, we outline a variety of CFPS platforms that provide alternative and complementary methods for expressing proteins from different organisms, compared with in vivo approaches...
October 14, 2016: Cold Spring Harbor Perspectives in Biology
Saravanan Krishnan, Shoba Narayan, Anju Chadha
The cell free extracts of Candida parapsilosis ATCC 7330 are more efficient than the whole resting cells of the yeast in the synthesis of directly usable gold nanoparticles as revealed by this systematic study. Cell free extracts yielded gold nanoparticles of hydrodynamic diameter (50-200 nm). In this study, the total protein concentration influences the nanofabrication and not only the reductase enzymes as originally thought. Powder X-ray diffraction studies confirm the crystalline nature of the gold nanoparticles...
December 2016: AMB Express
Eiko Seki, Natsuko Matsuda, Shigeyuki Yokoyama, Takanori Kigawa
No abstract text is available yet for this article.
October 3, 2016: Analytical Biochemistry
Chong-Jing Zhang, Jigang Wang, Jianbin Zhang, Yew Mun Lee, Guangxue Feng, Teck Kwang Lim, Han-Ming Shen, Qingsong Lin, Bin Liu
Understanding the mechanism of action (MOA) of bioactive natural products will guide endeavor to improve their cellular activities. Artemisinin and its derivatives inhibit cancer cell proliferation, yet with much lower efficiencies than their roles in killing malaria parasites. To improve their efficacies on cancer cells, we studied the MOA of artemisinin using chemical proteomics and found that free heme could directly activate artemisinin. We then designed and synthesized a derivative, ART-TPP, which is capable of targeting the drug to mitochondria where free heme is synthesized...
October 24, 2016: Angewandte Chemie
Stephen S Y Lam, Eric S K Ho, Bai-Liang He, Wui-Wing Wong, Chae-Yin Cher, Nelson K L Ng, Cheuk-Him Man, Harinder Gill, Alice M S Cheung, Ho-Wan Ip, Chi-Chiu So, Jerome Tamburini, Chi Wai Eric So, Dona N Ho, Chun-Hang Au, Tsun-Leung Chan, Edmond S K Ma, Raymond Liang, Yok-Lam Kwong, Anskar Y H Leung
An in vitro drug-screening platform on patient samples was developed and validated to design personalized treatment for relapsed/refractory acute myeloid leukemia (AML). Unbiased clustering and correlation showed that homoharringtonine (HHT), also known as omacetaxine mepesuccinate, exhibited preferential antileukemia effect against AML carrying internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD). It worked synergistically with FLT3 inhibitors to suppress leukemia growth in vitro and in xenograft mouse models...
October 5, 2016: Science Translational Medicine
Petra Bonova, Miroslava Nemethova, Milina Matiasova, Martin Bona, Miroslav Gottlieb
Ischemic tolerance (IT) has gained attention as an attractive strategy for improving stroke outcome. Recently, it was shown that signal responsible for rapid IT induction (tolerance induction factor- TIF) is transmitted via circulating blood. In the present paper, we have hypothesized about the role of the blood cell compartment in TIF production. We used hind limb ischemia to generate TIF as a rapid preconditioning against transient middle cerebral artery occlusion (MCAO). The essential properties of protein synthesis inhibitors actinomycin D and cycloheximide were utilized to obtain the following results: 1...
October 5, 2016: European Journal of Neuroscience
Y Q Jiang, X P Xu, Q M Guo, X C Xu, Q Y Liu, S H An, J L Xu, F Su, J B Tai
Drug resistance in cells is a major impedance to successful treatment of lung cancer. Taxus chinensis var. inhibits the growth of tumor cells and promotes the synthesis of interleukins 1 and 2 and tumor necrosis factor, enhancing immune function. In this study, T. chinensis var.-induced cell death was analyzed in lung cancer cells (H460) enriched for stem cell growth in a defined serum-free medium. Taxus-treated stem cells were also analyzed for Rhodamine 123 (Rh-123) expression by flow cytometry, and used as a standard functional indicator of MDR...
September 2, 2016: Genetics and Molecular Research: GMR
Kodai Machida, Tomoaki Shigeta, Ayano Kobayashi, Ai Masumoto, Yuna Hidaka, Hiroaki Imataka
Protein misfolding and aggregation is one of the major causes of neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Huntington's disease. So far protein aggregation related to these diseases has been studied using animals, cultured cells or purified proteins. In this study, we show that a newly synthesized polyglutamine protein implicated in Huntington's disease forms large aggregates in HeLa cells, and successfully recapitulate the process of this aggregation using a translation-based system derived from HeLa cell extracts...
October 1, 2016: Journal of Biotechnology
Yuqing Yin, Chen Ling Gao, Qi Xiao, Guo Lin, Zian Lin, Zongwei Cai, Huang-Hao Yang
Artificial enzyme mimetics have received considerable attention because natural enzymes have some significant drawbacks, including enzyme autolysis, low catalytic activity, poor recovery and low stability to environmental changes. Herein, we demonstrated a facile approach for one-pot synthesis of hemeprotein-metal organic framework hybrid composites (H-MOFs) by using bovine hemoglobin (BHb) and zeolitic imidazolate framework-8 (ZIF-8) as a model reaction system. Surprisingly, the new hybrid composites exhibits 423% increase in peroxidase-like catalytic activity compared to free BHb...
October 4, 2016: ACS Applied Materials & Interfaces
Lamia W Mohamed, Azza T Taher, Ghada S Rady, Mamdouh M Ali, Abeer E Mahmoud
A new series of benzothiazole has been synthesized as cytotoxic agents. The new derivatives were tested for their cytotoxic activity towards the human breast cancer MCF-7 cell line against cisplatin as the reference drug. Many derivatives revealed good cytotoxic effect, whereas four of them 4, 5c, 5d, 6b were more potent than cisplatin, with IC50 values being 8.64, 7.39, 7.56, 5.15 μM compared to 13.33 μM of cisplatin. The four derivatives cytotoxic activity was accompanied by regulating free radicals production, by increasing the activity of superoxide dismutase and depletion of intracellular reduced glutathione, catalase and glutathione peroxidase activities, accordingly, the high production of hydrogen peroxide, nitric oxide and other free radicals causing tumor cell death as monitored by reduction in the synthesis of protein and nucleic acids...
October 4, 2016: Chemical Biology & Drug Design
Chikkamenahalli Lakshminarayana Lakshmikanth, Shancy Petsel Jacob, Avinash Kundadka Kudva, Calivarathan Latchoumycandane, Puttaraju Srikanta Murthy Yashaswini, Mosale Seetharam Sumanth, Cassiano F Goncalves-de-Albuquerque, Adriana R Silva, Sridevi Annapurna Singh, Hugo C Castro-Faria-Neto, Sandeep Kumble Prabhu, Thomas M McIntyre, Gopal Kedihithlu Marathe
The endotoxin lipopolysaccharide (LPS) promotes sepsis, but bacterial peptides also promote inflammation leading to sepsis. We found, intraperitoneal administration of live or heat inactivated E. coli JE5505 lacking the abundant outer membrane protein, Braun lipoprotein (BLP), was less toxic than E. coli DH5α possessing BLP in Swiss albino mice. Injection of BLP free of LPS purified from E. coli DH5α induced massive infiltration of leukocytes in lungs and liver. BLP activated human polymorphonuclear cells (PMNs) ex vivo to adhere to denatured collagen in serum and polymyxin B independent fashion, a property distinct from LPS...
October 4, 2016: Scientific Reports
K Hartmann, J Bennien, B Wapelhorst, K Bakhaus, V Schumacher, S Kliesch, W Weidner, M Bergmann, J Geyer, D Fietz
Within the human testis, large amounts of sulfated steroid hormones are produced. As shown in breast tissue and placenta, these might not only be excretion intermediates, but re-activated in target cells by steroid sulfatase (STS). This process is called sulfatase pathway and may play a pivotal role in para- and/or intracrine regulation by creating a local supply for steroid hormones. This requires a facilitated transport via uptake carriers and efflux transporters as these hydrophilic molecules cannot pass the cell membrane...
September 29, 2016: Histochemistry and Cell Biology
Tuba Esatbeyoglu, Gerald Rimbach
The aim of this review is to summarize the relevant literature about the use of canthaxanthin in food science and nutrition research. Canthaxanthin is a red-orange carotenoid that belongs to the xanthophyll group. This naturally occurring pigment is present in bacteria, algae and some fungi. Canthaxanthin is also responsible for the color of flamingo feathers, koi carp skin and crustacean shells. Canthaxanthin is widely used in poultry (broiler, laying hens) as a feed additive. Canthaxanthin can be obtained by total synthesis...
September 30, 2016: Molecular Nutrition & Food Research
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