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Emma R Cold, Gerardo R Vasta, Josà A Fernà Ndez-Robledo
Perkinsus marinus is a protozoan parasite of molluscs that can be propagated in vitro in a defined culture medium, in the absence of host cells. We previously reported that P. marinus trophozoites can be transfected with high efficiency by electroporation using a plasmid based on MOE, a highly expressed gene, and proposed its potential use as a "pseudoparasite". This is a novel gene expression platform for parasites of medical relevance for which the choice of the surrogate organism is based on phylogenetic affinity to the parasite of interest, while taking advantage of the whole engineered surrogate organism as a vaccination adjuvant...
October 10, 2016: Journal of Parasitology
Qing Zhao, Wanghui Xu, Lei Xing, Zhanglin Lin
BACKGROUND: Peptides have recently become attractive for therapeutic applications. However, efficient production of medium- to large-sized peptides (30-100 amino acids [aa]) remains challenging both by recombinant and chemical synthesis. We previously reported the formation of active enzyme aggregates in Escherichia coli cells induced by the short β-structured peptide ELK16 (LELELKLKLELELKLK) and developed a streamlined protein expression and purification approach. In this approach, a cleavable self-aggregating tag (cSAT) consisting of an intein molecule and ELK16 was used to release the recombinant peptides with reasonable purity from active aggregates...
2016: Microbial Cell Factories
Jian Xu, Fei Zhong, Yonghong Zhang, Jianlou Zhang, Shanshan Huo, Hongyu Lin, Liyue Wang, Dan Cui, Xiujin Li
Probiotics and antimicrobial peptides (AMPs) in animal gastrointestinal tract play the crucial roles in microbiota maintenance, immune regulation and pathogen defending. Generating probiotics strain engineered for expressing AMPs should be beneficial for animal growth and production. Here, we generated B. subtilis strain engineered for expressing porcine β-defensin-2 (pBD-2) and cecropin P1 (CP1) fusion antimicrobial peptide using their codon-optimized genes mediated by pMK4 prokaryotic expression vector. Results showed that the B...
June 30, 2016: Asian-Australasian Journal of Animal Sciences
Han Jiang, Ping Li, Qing Gu
Bacteriocin, which is produced by lactic acid bacteria (LAB), has the potential to act as natural preservatives in the food industry. To develop strategies to overproduce such peptides, plantaricin NC8, a class IIb LAB bacteriocin that consists of two peptides, PLNC8α and PLNC8β, was successfully heterologously expressed in Escherichia coli BL21 (DE3). PLNC8α and PLNC8β peptides were expressed as His6-tag fusion proteins and were separated by Ni(2+) chelating affinity chromatography. To get the PLNC8α and PLNC8β peptides without extra amino acids in the N-terminus, the fusion proteins were cleaved by enterokinase and further purified using the Ni-NTA Sefinose™ Resin Kit...
November 2016: Protein Expression and Purification
John Moon, Juliette Gorson, Mary Elizabeth Wright, Laurel Yee, Samer Khawaja, Hye Young Shin, Yasmine Karma, Rajeeva Lochan Musunri, Michelle Yun, Mande Holford
Venom peptides found in terebrid snails expand the toolbox of active compounds that can be applied to investigate cellular physiology and can be further developed as future therapeutics. However, unlike other predatory organisms, such as snakes, terebrids produce very small quantities of venom, making it difficult to obtain sufficient amounts for biochemical characterization. Here, we describe the first recombinant expression and characterization of terebrid peptide, teretoxin Tgu6.1, from Terebra guttata. Tgu6...
March 2016: Toxins
Chunlan Xiao, Junyi Liu, Yanchun Tang, Junyong Chen, Xiaopeng Wu, Feng Bi, Jing Zhang
Nesfatin-1 is a newly discovered satiety molecule expressed mainly in the hypothalamic nuclei. It suppresses both short term and long term appetite. Six synthetic deoxyoligonucleotides overlapped by PCR encoding nesfatin-1 was cloned into a pET28a vector after the hexa-histidine tagged Multiple Cloning Sites sequence with an enterokinase recognition site incorporated in-between. The recombinant plasmid was transformed into E. coli strain Rosetta to express the fusion protein, which constituted 27% of the total cell proteins...
November 23, 2015: Biotechnology and Applied Biochemistry
Fanqiang Meng, Haizhen Zhao, Chong Zhang, Fengxia Lu, Xiaomei Bie, Zhaoxin Lu
A potential bacteriocin gene was isolated from 18575 ORFs by bioinformatics methods. It was named pln1, and cloned into pET32a. Then, it was expressed as a thioredoxin-Pln1 fusion protein in Escherichia coli BL21 (DE3). The fusion protein was purified by Ni-NTA, and thioredoxin was removed by enterokinase. Finally, Pln1 was purified using a cation affinity column. The yields of fused and cleaved Pln1 peptides were 100-110 mg/l and 9-11 mg/l, respectively. Pln1 was stable in an acidic environment and at temperatures below 60 °C, but was easily degraded under alkaline conditions and by protease treatment...
March 2016: Protein Expression and Purification
Mohamed Boumaiza, Maryse Jaouen, Jean-Christophe Deschemin, Aymen Ezzine, Noureddine Ben Khalaf, Sophie Vaulont, Mohamed Nèjib Marzouki, Marie Agnès Sari
Hepcidin, a 25-amino-acid and highly disulfide bonded antimicrobial peptide, is the central regulator of iron homeostasis. This hormone is expressed in response to iron and inflammation and interacts with ferroportin1 (FPN1), the only known iron exporter in vertebrates, inducing its internalization and degradation. Thus, the export of iron from cells to plasma will be significantly diminished. Thereby, hepcidin has become the target of intense research studies due to its profound biomedical significance. This study describes the functional expression of recombinant camel hepcidin in Escherichia coli...
November 2015: Protein Expression and Purification
Kai Qian, Chengyuan Li, Xiaohai Gong, Charles Ndawula, Jin Qian, Yun Chen, Huazhong Li, Jian Jin
OBJECTIVE: To improve the bioactivity and increase the N-terminal homogeneity of a glucagon-like peptide-1 (GLP-1) analogue expressed in Pichia pastoris. RESULTS: The GLP-1 analogue. GGH, consisting of two tandem mutant GLP-1 (GLP-1[A2G]) fused with the N-terminus of human serum albumin (HSA), was expressed in P. pastoris. We also designed and expressed the novel GLP-1 analogue NGGH, which had a His-tag fused with the N-terminus of GGH and an enterokinase (EK) cleavage site at the fusion junction...
November 2015: Biotechnology Letters
Hua Zhao, Jiayong Tang, Lei Cao, Gang Jia, Dingbiao Long, Guangmang Liu, Xiaoling Chen, Jingyi Cai, Haiying Shang
Parasin I (PI) is a 19 amino acid peptide with potent antimicrobial activities against a broad spectrum of microorganisms and is a good candidate for development as a novel antimicrobial agent. The objective of this study was to express and characterize a codon optimized parasin I peptide fused with human lysozyme (hLY). A 513 bp cDNA fragment encoding the mature hLY protein and parasin I peptide was designed and synthesized according to the codon bias of Pichia pastoris. A 4×Gly flexible amino acid linker with an enterokinase cleavage (DDDDK) was designed to link the PI to the C-terminal of hLY...
September 2015: Enzyme and Microbial Technology
Farokh Dotiwala, Isabelle Fellay, Luis Filgueira, Denis Martinvalet, Judy Lieberman, Michael Walch
When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY)...
2015: Journal of Visualized Experiments: JoVE
Gaurav Chhetri, Tripti Pandey, Ramesh Chinta, Awanish Kumar, Timir Tripathi
Amyloid-beta (Aβ) peptide mediates several neurodegenerative diseases. The 42 amino acid (Aβ1-42) is the predominant form of peptide found in the neuritic plaques and has been demonstrated to be neurotoxic in vivo and in vitro. The availability of large quantities of Aβ peptide will help in several biochemical and biophysical studies that may help in exploring the aggregation mechanism and toxicity of Aβ peptide. We report a convenient and economical method to obtain such a peptide biologically. Synthetic oligonucleotides encoding Aβ1-42 were constructed and amplified through the polymerase cycling assembly (also known as assembly PCR), followed by the amplification PCR...
October 2015: Protein Expression and Purification
Laure Bataille, Wilfrid Dieryck, Agnès Hocquellet, Charlotte Cabanne, Katell Bathany, Sébastien Lecommandoux, Bertrand Garbay, Elisabeth Garanger
Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. The recombinant expression of hydrophobic elastin-like polypeptides is often difficult because they possess low transition temperatures, and therefore form aggregates at sub-ambient temperatures. To circumvent this difficulty, we expressed in Escherichia coli three hydrophobic ELPs (VPGIG)n with variable lengths (n=20, 40, and 60) in fusion with the maltose-binding protein (MBP)...
June 2015: Protein Expression and Purification
Ana Rita Otrelo-Cardoso, Rashmi R Nair, Márcia A S Correia, Maria G Rivas, Teresa Santos-Silva
The TupABC system is involved in the cellular uptake of tungsten and belongs to the ABC (ATP binding cassette)-type transporter systems. The TupA component is a periplasmic protein that binds tungstate anions, which are then transported through the membrane by the TupB component using ATP hydrolysis as the energy source (the reaction catalyzed by the ModC component). We report the heterologous expression, purification, determination of affinity binding constants and crystallization of the Desulfovibrio alaskensis G20 TupA...
2014: International Journal of Molecular Sciences
Yuanzhi Cao, Feihua Yang, Weifeng Ma
OBJECTIVE: To construct a soluble prokaryotic expression vector of the CXCR7-specific antagonist SDF-1/54R and evaluate its activity. METHODS: SDF-1/54r gene amplified by PCR was inserted into the soluble expression vector pET-41a+ engineered with GST fusion tag, and the recombinant vector was transformed into E. coli strain BL21 (DE3). After IPTG induction of E. coli, the expressed recombinant protein was purified with GST affinity chromatography purification system and confirmed by SDS-PAGE and Western blotting assay...
June 2014: Nan Fang Yi Ke da Xue Xue Bao, Journal of Southern Medical University
M Lentze
With the rapid increase in knowledge on the genetic origin of diseases within the gastrointestinal tract the number of congenital diseases, which already manifest during childhood have drastically increased. Due to the large application of molecular genetics the number is steadily increasing. To make the access to these rare diseases fast and efficient the data base of the National Library of Medicine (Online Mendelian Inheritance of Man - OMIN) is a very helpful online tool, with which all these disease entities can be found easily (http://www...
May 2014: Georgian Medical News
Jing-juan Liang, Mei-ling Zhang, Meng Ding, Zhi-mao Mai, San-xing Wu, Yue Du, Jia-xun Feng
BACKGROUND: Kluyveromyces marxianus has recently become a species of interest for ethanol production since it can produce ethanol at high temperature and on a wide variety of substrates. However, the reason why this yeast can produce ethanol at high temperature is largely unknown. RESULTS: The ethanol fermentation capability of K. marxianus GX-UN120 at 40°С was found to be the same as that of Saccharomyces cerevisiae at 34°С. Zymogram analysis showed that alcohol dehydrogenase 1 (KmAdh1) was largely induced during ethanol production, KmAdh4 was constitutively expressed at a lower level and KmAdh2 and KmAdh3 were almost undetectable...
2014: BMC Biotechnology
Zdenko Levarski, Andrea Šoltýsová, Ján Krahulec, Stanislav Stuchlík, Ján Turňa
Human growth hormone (hGH) was one of the first recombinant proteins approved for the treatment of human growth disorders. Its small size (191 amino acids), possession of only 2 disulphide bonds and absence of posttranslational modifications make Escherichia coli the host of choice for its production on any scale. In this work, we have utilized an efficient T7 based expression system to produce high levels of soluble thioredoxin-hGH (Trx-hGH) fusion protein. We outline a relatively simple three step purification process employing two immobilized metal-affinity chromatography and one anion-exchange steps and removal of fusion partner by enterokinase cleavage yielding native hGH...
August 2014: Protein Expression and Purification
Jie Tao, Zhi Lei Zhou, Bin Wu, Jian Shi, Xiao Ming Chen, Yong Hua Ji
Martentoxin (MarTX), a 37-residue peptide purified from the venom of East-Asian scorpion (Buthus martensi Karsch), was capable of blocking large-conductance Ca2+-activated K+ (BK) channels. Here, we report an effective expression and purification approach for this toxin. The cDNA encoding martentoxin was expressed by the prokaryotic expression system pGEX-4T-3 which was added an enterokinase cleavage site by PCR. The fusion protein (GST-rMarTX) was digested by enterokinase to release hetero-expressed toxin and further purified via reverse-phase HPLC...
April 2014: Toxins
Yang Liu, Limei Ren, Lingmiao Ge, Qingxin Cui, Xiaofang Cao, Yuanyuan Hou, Fang Bai, Gang Bai
KGLP-1, a 31-amino acid glucagon-like peptide-1 (GLP-1) analogue, has a great therapeutic potential for anti-diabetes. In this work, a strategy for expression and purification of functional KGLP-1 peptide has been established. KGLP-1 cDNA was fused with glutathione S-transferase (GST), with an enterokinase cleavage site in the fusion junction. The recombinant fusion protein GST-KGLP-1 was affinity purified via the GST-tag, and then digested with enterokinase. The resulting GST part as well as the enzymes were eliminated by ultra-filtration followed by size exclusion chromatograph...
August 2014: Biotechnology Letters
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