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Maryam Raeeszadeh-Sarmazdeh, Ranganath Parthasarathy, Eric T Boder
Increasing interest in protein immobilization on surfaces has heightened the need for techniques enabling layer-by-layer protein attachment. Here, we report a technique for controlling enzyme-mediated immobilization of layers of protein on the surface using a genetically-encoded protecting group. An enterokinase-cleavable peptide sequence was inserted at the N-terminus of bifunctional fluorescent proteins containing Sortase A substrate recognition tags at both ends to control Sortase A-mediated protein immobilization on the surface layer-by-layer...
February 20, 2017: Biotechnology Progress
Leonel Vargas-Jaimes, Liang Xiao, Jing Zhang, Lourival D Possani, Héctor H Valdivia, Verónica Quintero-Hernández
BACKGROUND: Scorpion venoms contain toxins that modulate ionic channels, among which are the calcins, a small group of short, basic peptides with an Inhibitor Cystine Knot (ICK) motif that target calcium release channels/ryanodine receptors (RyRs) with high affinity and selectivity. Here we describe the heterologous expression of Intrepicalcin, identified by transcriptomic analysis of venomous glands from Vaejovis intrepidus. METHODS: Recombinant Intrepicalcin was obtained in Escherichia coli BL21-DE3 (periplasm) by fusing the Intrepicalcin gene to sequences coding for signal-peptide, thioredoxin, His-tag and enterokinase cleavage site...
April 2017: Biochimica et Biophysica Acta
J M Jiménez-Vargas, V Quintero-Hernández, L González-Morales, E Ortiz, L D Possani
This manuscript describes the design of plasmids containing the genes coding for four main mammalian toxins of scorpions from the genus Centruroides (C.) of Mexico. The genes that code for toxin 2 of C. noxius (Cn2), toxin 2 from C. suffusus (Css2) and toxins 1 and 2 from C. limpidus (Cll1 and Cll2) were included into individual plasmids carrying the genetic construction for expression of fusion proteins containing a leader peptide (pelB) that directs the expressed protein to the bacterial periplasm, a carrier protein (thioredoxin), the cleavage site for enterokinase, the chosen toxin and a poly-histidine tag (6xHis-tag) for purification of the hybrid protein by immobilized metal ion affinity chromatography after expression in Escherichia coli strain BL21 (DE3)...
March 15, 2017: Toxicon: Official Journal of the International Society on Toxinology
Marcela Pinedo, Melanie Genoula, María Ximena Silveyra, André De Oliveira Carvalho, Mariana Regente, Marianela Del Río, Júlia Ribeiro Soares, Valdirene Moreira Gomes, Laura De La Canal
According to their sugar recognition specificity, plant lectins are proposed as bioactive proteins with potential in cancer treatment and diagnosis. Helja is a mannose-specific jacalin-like lectin from sunflower which was shown to inhibit the growth of certain fungi. Here, we report its recombinant expression in a prokaryotic system and its activity in neurobalstoma cells. Helja coding sequence was fused to the pET-32 EK/LIC, the enterokinase/Ligation-independent cloning vector and a 35 kDa protein was obtained in Escherichia coli representing Helja coupled to thioredoxin (Trx)...
January 10, 2017: International Journal of Molecular Sciences
Jimin Pei, Nick V Grishin
SEA (sea urchin sperm protein, enterokinase, agrin) domains, many of which possess autoproteolysis activity, have been found in a number of cell surface and secreted proteins. Despite high sequence divergence, SEA domains were also proposed to be present in dystroglycan based on a conserved autoproteolysis motif and receptor-type protein phosphatase IA-2 based on structural similarity. The presence of a SEA domain adjacent to the transmembrane segment appears to be a recurring theme in quite a number of type I transmembrane proteins on the cell surface, such as MUC1, dystroglycan, IA-2, and Notch receptors...
March 2017: Protein Science: a Publication of the Protein Society
Kristína Melicherová, Ján Krahulec, Martin Šafránek, Veronika Lišková, Diana Hopková, Diana Széliová, Ján Turňa
Enterokinase is one of the most frequently used enzymes for the removal of affinity tags from target recombinant proteins. In this study, several fermentation strategies were assayed for the production of human enterokinase in Pichia pastoris under constitutive GAP promoter. Two of them with controlled specific growth rate during whole cultivation showed a very low enterokinase activity, under 1 U/ml, of the fermentation medium. On the contrary, the combined fermentation with a maximum specific growth rate at the initial phase of the fermentation and stationary-like phase during the rest of the fermentation showed a significant accumulation of the enterokinase in the medium, which counted up to 1400 U/ml...
March 2017: Applied Microbiology and Biotechnology
Emma R Cold, Gerardo R Vasta, José A Fernández 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...
February 2017: 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...
August 5, 2016: Microbial Cell Factories
Jian Xu, Fei Zhong, Yonghong Zhang, Jianlou Zhang, Shanshan Huo, Hongyu Lin, Liyue Wang, Dan Cui, Xiujin Li
OBJECTIVE: To generate recombinant Bacillus subtilis (B. subtilis) engineered for expression of porcine β-defensin-2 (pBD-2) and cecropin P1 (CP1) fusion antimicrobial peptide and investigate their anti-bacterial activity in vitro and their growth-promoting and disease resisting activity in vivo. METHODS: The pBD-2 and CP1 fused gene was synthesized using the main codons of B. subtilis and inserted into plasmid pMK4 vector to construct their expression vector. The fusion peptide-expressing B...
April 2017: 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 3, 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 were 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 Escherichia coli strain Rosetta to express the fusion protein, which constituted 27% of the total cell proteins...
January 2017: 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...
July 2, 2014: International Journal of Molecular Sciences
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