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Protein Expression and Purification

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https://www.readbyqxmd.com/read/28811266/expression-and-purification-of-a-difficult-sarcomeric-protein-telethonin
#1
Huanbo Tan, Wencheng Su, Pengju Wang, Wenyu Zhang, Michael Sattler, Peijian Zou
Telethonin anchors the N-terminal region of titin in the Z-disk of the sarcomere by binding to two immunoglobulin-like (Ig) domains (Z1 and Z2) of titin (Z1Z2). Thereby telethonin plays an important role in myofibril assembly and in muscle development and functional regulation. The expression and purification of recombinant telethonin is very challenging. In previous studies, recombinant telethonin expressed from E. coli was refolded in the presence of Z1Z2. Here, we report various strategies to establish a reliable and efficient protocol for the preparation of telethonin and titin Z1Z2 protein...
August 12, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28811265/molecular-cloning-heterologous-expression-and-functional-characterization-of-gamma-tocopherol-methyl-transferase-%C3%AE-tmt-from-glycine-max
#2
Kalpana Tewari, Anil Dahuja, Archana Sachdev, Kishwar Ali, Vaibhav Kumar, Amresh Kumar, Sweta Kumari
γ-Tocopherol methyltransferase (γ-TMT) (EC 2.1.1.95) is the last enzyme in the tocopherol biosynthetic pathway and it catalyzes the conversion of γ-tocopherol into α-tocopherol, the nutritionally significant and most bioactive form of vitamin E. Although the γ-TMT gene has been successfully overexpressed in many crops to enhance their α-tocopherol content but still only few attempts have been made to uncover its structural, functional and regulation aspects at protein level. In this study, we have cloned the complete 909bp coding sequence of Glycine max γ-TMT (Gm γ-TMT) gene that encodes the corresponding protein comprising of 302 amino acid residues...
August 12, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28807840/the-heterologous-expression-strategies-of-antimicrobial-peptides-in-microbial-systems
#3
REVIEW
Ting Deng, Haoran Ge, Huahua He, Yao Liu, Chao Zhai, Liang Feng, Li Yi
Antimicrobial peptides (AMPs) consist of molecules acting on the defense systems of numerous organisms toward tumor and multiple pathogens, such as bacteria, fungi, viruses, and parasites. Compared to traditional antibiotics, AMPs are more stable and have lower propensity for developing resistance through functioning in the innate immune system, thus having important applications in the fields of medicine, food and so on. However, despite of their high economic values, the low yield and the cumbersome extraction process in AMPs production are problems that limit their industrial application and scientific research...
August 11, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28807839/characterization-of-fm2382-from-fulvimarina-manganoxydans-sp-nov-8047-with-potential-enzymatic-decontamination-of-sulfur-mustard
#4
Yuan-Zhong Zhao, Xuan Guo, Jin-Yi Zhong, Nan Guo, Lin-Cai Chen, Zhi-Yang Dong
Sulfur mustard (SM) can be hydrolyzed by haloalkane dehalogenases such as DhaA, LinB and DmbA. However, the low resistance to the elevated temperatures limited the practical application of haloalkane dehalogenases. Here we reported a new thermotolerant dehalogenase FM2382 from Fulvimarina manganoxydans sp. nov. 8047. The specific activity of FM2382 to SM is 0.6 U/mg. FM2382 possessed high heat stability (45 °C) in slight alkali environment (pH 7.5) and retained approximately 50% activity after incubation at 70 °C for 40 min...
August 11, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28802644/rapid-characterization-of-the-cho-platform-cell-line-and-identification-of-pseudo-attp-sites-for-phic31-integrase
#5
Narges Damavandi, Mozhgan Raigani, Atefeh Joudaki, Fatemeh Davami, Sirous Zeinali
The Chinese Hamster Ovary (CHO) cell lines, applicable to post-translational modifications, are preferred systems for biopharmaceutical protein production. In this study, by using the Jump-In™ TI™ technology which employs PhiC31 and R4 bacteriophage recombinases, a platform CHO-K1 cell line containing a R4-attP site was generated. Here, a combination of Quantitative Fluorescent-Polymerase Chain Reaction (QF-PCR) and semi-random, two-step PCR (ST-PCR), was performed to feature the platform cell clones. Our results show that QF-PCR and ST-PCR, can be utilized for efficient and accelerated cell line characterization...
August 9, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28757468/identification-cloning-and-expression-analysis-of-an-alpha-cgtase-produced-by-stain-y112
#6
Jian-Hua Hao, Li-Ping Huang, Xiao-Tong Chen, Jing-Jing Sun, Jun-Zhong Liu, Wei Wang, Mi Sun
Cyclodextrin glycosyltransferase (CGTase) is an enzyme able to convert starch and other substrates into cyclodextrins (CDs). A marine strain Y112 producing α-CGTase was identified as Bacillus agaradhaerens Y112 by physiological and biochemical characterization, and 16S rDNA analysis. The gene coding for α-CGTase was cloned, sequenced and expressed in Escherichia coli BL21 (DE3) cells. Recombinant α-CGTase was purified in one-step chromatographic separation and its purity evaluated by SDS-PAGE, showing the presence of one band with a molecular mass of about 92 kDa...
July 27, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28757467/high-level-expression-and-purification-of-soluble-form-of-human-natural-killer-cell-receptor-nkr-p1-in-hek293s-gnti-cells
#7
Jan Bláha, Barbora Kalousková, Ondřej Skořepa, Samuel Pažický, Petr Novák, Ondřej Vaněk
Human natural killer receptor protein 1 (NKR-P1, CD161, gene klrb1) is a C-type lectin-like receptor of natural killer (NK) cells responsible for recognition of its cognate protein ligand lectin-like transcript 1 (LLT1). NKR-P1 is the single human orthologue of the prototypical rodent NKR-P1 receptors. Naturally, human NKR-P1 is expressed on the surface of NK cells, where it serves as inhibitory receptor; and on T and NKT cells functioning as co-stimulatory receptor promoting secretion of IFNγ. Most notably, it is expressed on Th17 and Tc17 lymphocytes where presumably promotes targeting into LLT1 expressing immunologically privileged niches...
July 27, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28751017/co-refolding-of-a-functional-complex-of-dengue-ns3-protease-and-ns2b-co-factor-domain-and-backbone-resonance-assignment-by-solution-nmr
#8
Esmeralda Woestenenk, Peter Agback, Sofia Unnerståle, Ian Henderson, Tatiana Agback
A novel approach for separate expression of dengue virus NS3 protease and its NS2B cofactor domain is described in this paper. The two proteins are expressed in E.coli and purified separately and subsequently efficiently co-refolded to form a stable complex. This straightforward and robust method allows for separate isotope labeling of the two proteins, facilitating analysis by nuclear magnetic resonance (NMR) spectroscopy. Unlinked NS2B-NS3pro behaves better in NMR spectroscopy than linked NS2B-NS3pro, which has resulted in the backbone resonance assignment of the unlinked NS2B-NS3 complex bound to a peptidic boronic acid inhibitor...
July 24, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28736314/expression-and-purification-of-single-cysteine-containing-mutant-variants-of-the-mouse-prion-protein-by-oxidative-refolding
#9
Ishita Sengupta, Jayant B Udgaonkar
The folding and aggregation of proteins has been studied extensively, using multiple probes. To facilitate such experiments, introduction of spectroscopically-active moieties in to the protein of interest is often necessary. This is commonly achieved by specifically labelling cysteine residues in the protein, which are either present naturally or introduced artificially by site-directed mutagenesis. In the case of the recombinant prion protein, which is normally expressed in inclusion bodies, the presence of the native disulfide bond complicates the correct refolding of single cysteine-containing mutant variants of the protein...
July 21, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28734840/an-improved-purification-method-for-the-lysosomal-storage-disease-protein-%C3%AE-glucuronidase-produced-in-cho-cells
#10
Erica J Fratz-Berilla, Stephanie A Ketcham, Hamideh Parhiz, Muhammad Ashraf, Chikkathur N Madhavarao
Human β-glucuronidase (GUS; EC 3.2.1.31) is a lysosomal enzyme that catalyzes the hydrolysis of β-d-glucuronic acid residues from the non-reducing termini of glycosaminoglycans. Impairment in GUS function leads to the metabolic disorder mucopolysaccharidosis type VII, also known as Sly syndrome. We produced GUS from a CHO cell line grown in suspension in a 15 L perfused bioreactor and developed a three step purification procedure that yields ∼99% pure enzyme with a recovery of more than 40%. The method can be completed in two days and has the potential to be integrated into a continuous manufacturing scheme...
July 19, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28734839/recombinant-expression-of-ixolaris-a-kunitz-type-inhibitor-from-the-tick-salivary-gland-for-nmr-studies
#11
V S De Paula, F H S Silva, I M B Francischetti, R Q Monteiro, A P Valente
Ixolaris is an anticoagulant protein identified in the tick saliva of Ixodes scapularis. Ixolaris contains 2 Kunitz like domains and binds to Factor Xa or Factor X as a scaffold for inhibition of the Tissue Factor (TF)/Factor VIIa (FVIIa). In contrast to tissue factor pathway inhibitor (TFPI), however, Ixolaris does not bind to the active site cleft of FXa. Instead, complex formation is mediated by the FXa heparin-binding exosite. Due to its potent and long-lasting antithrombotic activity, Ixolaris is a promising agent for anticoagulant therapy...
July 19, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28734838/expression-purification-and-characterization-of-sll1981-protein-from-cyanobacterium-synechocystis-sp-pcc6803
#12
Xiaoqin Wang, Guofeng Lei, Xiaoyu Wu, Fei Wang, Chongde Lai, Zhimin Li
The sll1981 protein from cyanobacterium Synechocystis sp. PCC6803 had been reported to exhibit acetolactate synthase (ALS) and L-myo-inositol-1-phosphate synthase (MIPS) activities previously. Based on amino acids sequences alignment, sll1981 protein was postulated to function as α-ketoglutarate decarboxylase (α-KGD), which played important role in completing cyanobacterial tricarboxylic acid (TCA) cycle. However the detailed enzymatic kinetics of sll1981 as ALS, MIPS and α-KGD were not determined yet. In this study, the recombinant sll1981 protein was purified from supernatant of E...
July 19, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28732682/characterization-and-purification-of-proteins-suitable-for-the-production-of-antibodies-against-ca-liberibacter-asiaticus
#13
Huawei Liu, Sagheer Atta, John S Hartung
The citrus disease huanglongbing (HLB), which is caused by 'Candidatus Liberibacter asiaticus' (CaLas), is one of the most devastating pathogens of citrus, and with no effective method of control, poses a serious threat to citrus production throughout the world. In a previous study we described the production of single chain antibodies against several CaLas proteins that provide the basis for efficient and accurate detection of CaLas in citrus tissues. The isolation of a sufficient amount of purified antigen is a key step in the production of functional antibodies...
July 18, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28728943/production-and-biophysical-characterization-of-a-mini-membrane-protein-ost4v23d-a-functionally-important-mutant-of-yeast-oligosaccharyltransferase-subunit-ost4p
#14
Bharat Chaudhary, Suman Mazumder, Smita Mohanty
N-linked glycosylation of proteins is an essential and highly conserved co- and post-translational protein modification reaction that occurs in all eukaryotes. Oligosaccharyltransferase (OST), a multi-subunit membrane-associated enzyme complex, carries out this reaction. In the central reaction, a carbohydrate group is transferred to the side chain of a consensus asparagine residue in the newly synthesized protein. Genetic defects in humans cause a series of disorders known as congenital disorders of glycosylation (CDG) that include mental retardation, developmental delay, hypoglycemia etc...
July 18, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28712957/secretory-expression-of-%C3%AE-mannanase-from-bacillus-circulans-nt-6-7-in-lactobacillus-plantarum
#15
Kwankanit Intaratrakul, Sunee Nitisinprasert, Thu-Ha Nguyen, Dietmar Haltrich, Suttipun Keawsompong
The β-mannanase gene of Bacillus circulans NT 6.7 was successfully cloned in Lactobacillus plantarum WCFS1 using the pSIP403 expression vector and secreted to the supernatant rather than accumulated in the cells. The highest activity was achieved by controlling the pH at 6 during cultivation. Maximum mannanase activities detected in the supernatant and cell-free extract of 200 ml MRS broth were 8.2 and 0.86 U/ml, respectively. Enzyme activity in the supernatant increased to 27 U/ml by fermentation in a 5-L bioreactor with automatic pH control...
July 13, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28712956/refolding-of-a-novel-cholesterol-oxidase-from-pimelobacter-simplex-reveals-dehydrogenation-activity
#16
Hui-Min Qin, Jian-Wen Wang, Qianqian Guo, Songtao Li, Panpan Xu, Zhangliang Zhu, Dengyue Sun, Fuping Lu
Cholesterol oxidases, which catalyze the degradation of cholesterol to cholest-4-en-3-one, are widely used in the pharmaceutical and food processing industries. The cholesterol oxidase from Pimelobacter simplex (PsChO3) was transformed into E. coli BL21(DE3), but it was expressed mainly as inclusion bodies, and any soluble PsChO3 failed to bind to Ni-NTA resin. To overcome this obstacle, we devised a simple yet efficient purification and refolding process using 8 M urea for the solubilization of PsChO3 and achieved a high yield of the enzyme in its active form...
July 13, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28711732/high-level-expression-and-purification-of-a-mollusca-endoglucanase-from-ampullaria-crossean-in-pichia-pastoris
#17
Xiuping Su, Xinwei Geng, Meixian Fu, Yeqing Wu, Longfei Yin, Fukun Zhao, Wei Chen
EG27I is an endogenous glucanase belonging to glycoside hydrolase family (GHF) 45 from the Mollusca, Ampullaria crossean. In this study, the EG27I mature peptide gene fused to HFBII secretion signal of Trichoderma reesei was expressed under GAP promoter of Pichia pastoris in SMD1163 strain. Bioactive EG27I with a molecular weight of 27 kDa was successfully expressed and secreted into the culture medium. When high cell density fermentation of the recombinant Pichia pastoris was performed by a fed-batch strategy for totally 132 h in a 7...
July 12, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28711730/purification-and-characterization-of-a-long-acting-ciliary-neurotrophic-factor-via-genetically-fused-with-an-albumin-binding-domain
#18
Longfu Xu, Chun Zhang, Liping Liu, Yao Zhang, Qi Wang, Jian Wang, Yongdong Liu, Zhiguo Su
Ciliary neurotrophic factor (CNTF) is a promising candidate for the treatment of neurodegenerative or metabolic diseases, but suffers rapid clearance in body. Herein we constructed a new long-acting recombinant human CNTF (rhCNTF) by genetic fusion with an albumin-binding domain (ABD) through a flexible peptide linker, hoping to endow the new molecule prolonged serum circulation time by binding with endogenous human serum albumin (HSA) and then utilizing the naturally long-half-life property of HSA. This fused protein rhCNTF-ABD was expressed in Escherichia coli mainly in the soluble form and purified through a two-step chromatography, with purity of 95% and a high yield of 90-100 mg/L culture...
July 12, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28602686/heterologous-expression-in-pichia-pastoris-and-characterization-of-a-novel-gh11-xylanase-from-saline-alkali-soil-with-excellent-tolerance-to-high-ph-high-salt-concentrations-and-ethanol
#19
REVIEW
Hui Wang, Zhongyuan Li, Huihui Liu, Shuang Li, Haiyan Qiu, Kun Li, Xuegang Luo, Yajian Song, Nan Wang, Hongpeng He, Hao Zhou, Wenjian Ma, Tongcun Zhang
A GH11 xylanase gene (xyn11-1) cloned from saline-alkali soil was successfully expressed in Pichia pastoris GS115. The purified recombinant Xyn11-1 showed its maximal activity at pH 6.0, and retained more than 60.4% of activity at pH 10.0, with good pH stability. Its optimal temperature was 50 °C and it was stable after incubation for 1 h at 30 °C. Furthermore, Xyn11-1 was highly salt-tolerant, retaining more than 77.4% of activity in the presence of 0.25-4 M NaCl and displaying more than 47.2% relative activity after being incubated in the presence of 5 M NaCl at 37 °C for 10 min...
June 7, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28711733/expression-and-purification-of-tat-ndrg2-recombinant-protein-and-evaluation-of-its-anti-proliferative-effect-on-lncap-cell-line
#20
Fahimeh Farokhinejad, Abbas Behzad Behbahani, Gholam Reza Rafiei Dehbidi, Mohammad Ali Takhshid
N-myc downstream regulated gene2 (NDRG2) belongs to tumor suppressor protein family of NDRG. Anti-proliferative and anti-metastasis of NDRG2 overexpression has been demonstrated in a number of tumors. The aim of this study was to fuse the gene of Trans Activator of Transcription (TAT) protein transduction domain with NDRG2 gene and express and purify TAT-NDRG2 fusion protein in order to investigate the effects of TAT-NDRG2 protein on proliferation and apoptosis of LNCaP prostate carcinoma cell line. pET28a-TAT-NDRG2 and pET28a-NDRG2 plasmids were constructed and transformed into E...
October 2017: Protein Expression and Purification
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