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Protein Expression and Purification

Thanunthon Bowornsakulwong, Walaiporn Charoensapsri, Triwit Rattanarojpong, Pongsak Khunrae
WSSV134 or VP36A protein of white spot syndrome virus was previously reported to be able to reduce apoptosis in Sf-9 cells transfected with caspase of Penaeus monodon (PmCasp). The protein was therefore believed to have a role in supporting the survival of WSSV inside the host cells during infection. However, the anti-apoptosis activity of WSSV134 involved in the inhibition of PmCasp is still unclear. In this study, we produced a recombinant WSSV134 (rWSSV134) and tested for its ability to inhibit PmCasp in vitro...
October 15, 2016: Protein Expression and Purification
Andrés Corral Lugo, Abdelali Daddaoua, Alvaro Ortega, Bertrand Morel, Ana Isabel Díez Peña, Manuel Espinosa-Urgel, Tino Krell
Quorum sensing systems are essential for bacterial communication. We report here the purification and characterization of the Pseudomonas aeruginosa LasR quorum sensing regulator purified from lysates of E. coli cultures grown in the absence of added acyl-homoserine lactones (AHL). We show by isothermal titration calorimetry that LasR recognizes different AHLs with an affinity of approximately 1 μM. The affinity of LasR for its cognate 3-Oxo-C12-AHL was similar to that of other AHLs, indicating that this regulator has not evolved to preferentially recognize its cognate AHL...
October 15, 2016: Protein Expression and Purification
Youran Li, Zhenghua Gu, Liang Zhang, Zhongyang Ding, Guiyang Shi
Trehalose synthase (TreS) could transform maltose into trehalose via isomerization. It is a crucial enzyme in the process of trehalose enzymatical transformation. In this study, plasmid-based inducible expression systems were constructed to produce Thermomonospora curvata TreS in B. licheniformis. Xylose operons from B. subtilis, B. licheniformis and B. megaterium were introduced to regulate the expression of the gene encoding TreS. It was functionally expressed, and the BlsTs construct yielded the highest enzyme activity (12...
October 14, 2016: Protein Expression and Purification
Jakub Gabrielczyk, Jonas Kluitmann, Thorben Dammeyer, Hans-Joachim Jördening
For commercial applications refolding process must be fast, inexpensive and highly efficient. In the past many strategies for protein refolding were introduced. Still, simple refolding methods with high product concentrations are still rare. Refolding experiments were performed with fructosyltransferase (FTF, EC from Bacillus subtilis NCIMB 11871 produced as inclusion bodies. Solubilizates were refolded with batch dialysis or by continuous exchange of dialysis buffers with variable ionic strength...
October 14, 2016: Protein Expression and Purification
De-Mei Meng, Jing-Fang Zhao, Xiao Ling, Hong-Xia Dai, Ya-Jun Guo, Xiao-Fang Gao, Bin Dong, Zi-Qi Zhang, Xin Meng, Zhen-Chuan Fan
The antimicrobial peptide PaDef was isolated from Mexican avocado fruit and was reported to inhibit the growth of Escherichia coli and Staphylococcus aureus in 2013. In this study, an N-terminal 6 × His tagged recombinant PaDef (rPaDef) with a molecular weight of 7.5 KDa, for the first time, was expressed as a secreted peptide in Pichia pastoris. The optimal culture condition for rPaDef expression was determined to be incubation with 1.5% methanol for 72 h at 28 °C under pH 6.0. Under this condition, the amount of the rPaDef accumulation reached as high as 79...
October 11, 2016: Protein Expression and Purification
Ming Liu, Li-Na Li, Yi-Ting Pan, Jian-Qiang Kong
As the first step of ongoing efforts to investigate the genes responsible for the biosynthesis of steroidal saponins in the medicinal plant Ornithogalum caudatum, this investigation reported the cDNA isolation, prokaryotic expression and functional characterization of squalene synthase (SQS) gene from O. caudatum for the first time. Specifically, two unigenes showing high sequence identity to SQS were retrieved from RNA-Taq data, and then a full-length OcSQS1 corresponding to the two unigenes was isolated from O...
October 7, 2016: Protein Expression and Purification
Maria C Bewley, Lisa Reinhart, Matthew S Stake, Shorena Nadaraia-Hoke, Leslie J Parent, John M Flanagan
HIV Gag, a multidomain polyprotein that orchestrates the assembly and release of the human immunodeficiency virus (HIV), is an active target of antiretroviral inhibitor development. However, highly pure, stable, recombinant Gag has been difficult to produce in quantities sufficient for biophysical studies due to its susceptibility to proteolysis by cellular proteases during purification. Stability has been improved by using a construct that omits the p6 domain (deltap6Gag). Since in vivo, p6 is crucial to the budding process and interacts with protein complexes in the ESCRT pathway, it has been difficult to study its role the context of Gag using in vitro approaches...
October 6, 2016: Protein Expression and Purification
Steen Sørensen, Vibeke Myrhøj, Thanh Ha Nguyen, Per Aaslo, Young Bae Hansen
INTRODUCTION: Glycodelin is a glycoprotein with different oligosaccharides that are responsible for its diverse biological functions in contraception and immunosuppression. Therefore, it is necessary to have access to adequate amounts of glycodelin with retained carbohydrate structure for functional studies because the carbohydrate part can be lacking or be insufficient in recombinant glycodelin from prokaryotic and eukaryotic cell systems. METHODS AND RESULTS: Native glycodelin was purified from amniotic fluid by a series of affinity chromatography steps and had many glycosylated forms verified by mass spectrometry...
October 3, 2016: Protein Expression and Purification
M A Shulepko, E N Lyukmanova, Z O Shenkarev, P V Dubovskii, M V Astapova, A V Feofanov, A S Arseniev, Y N Utkin, M P Kirpichnikov, D A Dolgikh
Cytotoxins or cardiotoxins is a group of polycationic toxins from cobra venom belonging to the 'three-finger' protein superfamily (Ly6/uPAR family) which includes small β-structural proteins (60-90 residues) with high disulfide bond content (4-5 disulfides). Due to a high cytotoxic activity for cancer cells, cytotoxins are considered as potential anticancer agents. Development of the high-throughput production methods is required for the prospective applications of cytotoxins. Here, efficient approach for bacterial production of recombinant analogue of cytotoxin I from N...
October 1, 2016: Protein Expression and Purification
Yanqing Li, Ya Li, Mingfu Ye, Dongyang Wang, Junli Zhao, Xiaohong Sun, Qinwen Mao, Haibin Xia
Progranulin (PGRN), a highly glycosylated, secreted 593 amino acid precursor protein, is a multifunctional molecule that is critical for early embryogenesis, wound repair, inflammatory and tumorigenesis. PGRN can be proteolytically cleaved into seven cysteine-rich granulin (Grn) peptides: G, F, B, A, C, D and E. Both PGRN and its constituent Grn peptides have been implicated in a wide variety of biological activities. However, their functions are far from clear, and the lack of granulin domain-specific antibodies has hindered the progress of the functional study of PGRN and Grns...
September 29, 2016: Protein Expression and Purification
Bianca S Siedler, Bárbara C Roloff, Gizele L de Sá, Alessandra Neis, Fabrício R Conceição, Daiane D Hartwig, Sibele Borsuk, Odir A Dellagostin, Fabrício S Campos, Paulo M Roehe, Claudia P Hartleben, Alan J A McBride
Bovine herpesvirus (BoHV) glycoprotein E (gE) is a non-essential envelope glycoprotein and the deletion of gE has been used to develop BoHV-1 and BoHV-5 differential vaccine strains. The DIVA (Differentiation of Infected from Vaccinated Animals) strategy, using marker vaccines based on gE-negative BoHV strains, allows the identification of vaccinated or infected animals in immunoassays designed to detect anti-gE antibodies. In this study a codon optimized synthetic sequence of gE containing highly conserved regions from BoHV-1 and BoHV-5 was expressed in Pichia pastoris...
September 28, 2016: Protein Expression and Purification
Lu Fu, Hao Jin, Yongjiao Yu, Bin Yu, Haihong Zhang, Jiaxin Wu, Yuhe Yin, Xianghui Yu, Hui Wu, Wei Kong
The Norovirus (NoV) P domain, with three surface loops for foreign antigen insertion, has been demonstrated as an excellent platform for antigen presentation and novel vaccine development. The P domain alone can self-assemble into a P dimer, 12-mer small particle or 24-mer P particle, and vaccines based on those particles may elicit different levels of immunogenicity. Currently, P particles are generally produced in soluble expression systems in Escherichia coli, mainly in the 24-mer form. However, the low yield of the soluble protein has hindered further clinical applications of P particle-based protein vaccines...
September 28, 2016: Protein Expression and Purification
Muthusamy Sanjivkumar, TamilSelvan Silambarasan, Arunachalam Palavesam, Grasian Immanuel
The present study was undertaken to evaluate the xylanolytic properties of an actinobacterium Streptomyces olivaceus (MSU3) isolated from the sediment sample of mangrove environment. It showed highest xylanase activity on initial screening in Breg's mineral salts medium supplemented with 0.5% xylan. Further the organism expressed maximum xylanase production at optimized culture conditions of pH 7, temperature 30 °C with 2.5% of inoculum size at 72 h of incubation, the nutrient sources like sucrose (2%) and yeast extract (3%) have observed as best carbon and nitrogen sources respectively...
September 28, 2016: Protein Expression and Purification
Xiu-Li Cao, Xing Zhang, Yu-Fei Zhang, Yi-Zhe Zhang, Chang-Geng Song, Fan Liu, Yi-Yang Hu, Min-Hua Zheng, Hua Han
Ttyh1 is a murine homolog of the Drosophila Tweety and is predicted as a five-pass transmembrane protein. The Ttyh1 mRNA is expressed in mouse brain tissues with a restricted pattern and in human glioma cells. Ttyh1 protein may function as a large-conductance chloride channel, however, the role of Ttyh1 in normal neural development and tumorigenesis has been largely unknown, at least partially due to the lack of effective antibodies. Here we report the expression in E. coli and purification of two recombinant Ttyh1 protein fragments corresponding to one of the predicted extracellular domains and the carboxyl terminus of the mouse Ttyh1...
September 24, 2016: Protein Expression and Purification
Thomas K Karikari, Alexandra Turner, Robert Stass, Leonie C Y Lee, Bethany Wilson, David A Nagel, Eric J Hill, Kevin G Moffat
Recombinant tau protein is widely used to study the biochemical, cellular and pathological aspects of tauopathies, including Alzheimer's disease and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTPD-17). Pure tau in high yield is a requirement for in vitro evaluation of the protein's physiological and toxic functions. However, the preparation of recombinant tau is complicated by the protein's propensity to aggregate and form truncation products, necessitating the use of multiple, time-consuming purification methods...
September 20, 2016: Protein Expression and Purification
Ebru Kocadag Kocazorbaz, Figen Zihnioglu
Prolyl oligopeptidase (POP, EC is a cytosolic serine protease that hydrolyses proline containing small peptides. The members of prolyl oligopeptidase family play important roles in many physiological processes such as neurodegenerative diseases, maturation and degradation of peptide hormones. Thus the enzyme has been purified and characterized from various sources to elucidate the potential use as therapeutics. In this study recombinant Myxococcus xanthus prolyl oligopeptidase expressed in E. coli was purified 60...
January 2017: Protein Expression and Purification
Shahryar Khoshtinat Nikkhoi, Fatemeh Rahbarizadeh, Davoud Ahmadvand
Variable heavy chain of HcAb (VHH), the smallest intact antibody fragment, possesses sub-nanomolar affinity to antigens. In spite of conventional antibodies, these fragments recognize concave and linear epitopes. VHHs are one the best weapon for targeted drug delivery in nanomedicine and biopharmaceutics. HER2 is overexpressed in 20-25% of breast and ovarian cancers. For many reasons, HER2 is a prominent target for drug delivery to breast tumor. In this study, we designed a robust prokaryotic expression system to express functional VHHs against HER2 receptor...
January 2017: Protein Expression and Purification
Wang YaPing, Rao Ben, Yan Hong, Han Rui, Li Li, Liao Ping'an, Ma Lixin
l-glutamate oxidase (GLOD), encoded by the gox gene, catalyses the transformation of l-glutamic acid into α-ketoglutaric acid (α-KG). In the present study, Pichia pastoris was used for heterologous production of GLOD following optimization of the gox coding sequence for expression in the yeast host. A series of constructs based on the pHBM905BDM plasmid were engineered and transformed into P. pastoris to increase the gox copy number. The results indicated that GLOD protein levels and enzyme activity increased with increasing gox copy number...
January 2017: Protein Expression and Purification
Mahdi Abbasian, Hadieh Alsadat Eslampanah Seyedi, Badraldin Ebrahim Sayed Tabatabaei, Zahra Arab-Bafrani, Mohammad Reza Mofid, Reza Zareie
Vessel dilator is a 3.9-KDa potent anticancer peptide and a valuable candidate in the treatment of conditions such as congestive heart failure and acute renal failure amongst others. Here we report the recombinant production of vessel dilator in Escherichia coli. Three different synthetic ORF's dubbed VDI, VDII and VDIII, each encoding a trimmer of the vessel dilator peptide attached to a His tag sequence at their C- terminal, were synthesized and placed in pET21c expression vectors. The highest yield, following expression in E...
January 2017: Protein Expression and Purification
Yanbing Cui, Yiwei Meng, Juan Zhang, Bin Cheng, Huijia Yin, Chao Gao, Ping Xu, Chunyu Yang
In well-established heterologous hosts, such as Escherichia coli, recombinant proteins are usually intracellular and frequently found as inclusion bodies-especially proteins possessing high rare codon content. In this study, successful secretory expression of three hydrolases, in a constructed inducible or constitutive system, was achieved by fusion with a novel signal peptide (Kp-SP) from an actinomycete. The signal peptide efficiently enabled extracellular protein secretion and also contributed to the active expression of the intracellular recombinant proteins...
January 2017: Protein Expression and Purification
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