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Protein Expression and Purification

Chuan Zhang, An Li, Ruina Wang, Yan Cao, Han Jiang, Songying Ouyang, Xingyuan Ma, Yiming Lu
The interaction between TNF-α with TNFR1 triggers important signaling pathways inducing diverse cellular phenomena including inflammation, apoptosis, etc., and is involved in the pathogenesis and progression of numerous autoimmune diseases. The extracellular domain (ECD) of TNFR has been successfully used to clinically treat such TNF-associated diseases. However, large-scale production of these biological material via eukaryotic cell expression systems is usually costly owing to the culture medium and complicated growth conditions...
November 8, 2018: Protein Expression and Purification
Quan Chen, Yuhang Zhou, Jianli Yu, Wenshuai Liu, Fei Li, Mo Xian, Rui Nian, Haipeng Song, Dongxiao Feng
Nanobodies offer multiple advantages over conventional antibodies in terms of size, stability, solubility, immunogenicity, and production costs, with improved tumor uptake and blood clearance. Additionally, the recombinant expression of nanobodies is robust in various expression systems, such as Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris. P. pastoris is the most widely used microorganism for nanobody production, but all or almost all expression vectors developed for this system are based on the regulated promoter of the alcohol oxidase 1 gene (AOX1) that requires methanol for full induction...
November 8, 2018: Protein Expression and Purification
Jian-Yong Zheng, Xing Lan, Xiao-Jun Li, Li-Juan Huang, Yin-Jun Zhang, Zhao Wang
Pichia pastoris expression is a mature and efficient eukaryotic expression system. In this work, Aspergillus oryzae lipase (AOL, with the molecular mass of 28 kDa), which can perform highly stereoselective hydrolysis of (R, S)-methyl 2-(4-hydroxyphenoxy) propanoate, was expressed in P. pastoris X-33. The specific activity of AOL was 432 U/mg, which was obtained by fed-batch cultivation in a 5 L bioreactor using a methanol feeding strategy. After fermentation, the supernatant was concentrated by ultrafiltration with a 10 kDa cut-off membrane and purified with DEAE-Sepharose™ FF ion-exchange chromatography and phenyl Seflnose™ 6 FF hydrophobic interaction chromatography...
October 31, 2018: Protein Expression and Purification
Seyedeh Roghayeh Hamidi, Yaghoub Safdari, Mehdi Sheikh Arabi
One of a major drawbacks correlated with expressing antibody fragments in bacterial cells is insolubility, which is often regarded as an obstacle in obtaining active molecules. Recombinant proteins aggregated as inclusion bodies within bacterial cells are thought to be unfolded or misfolded, and therefore inactive. So, denaturing and refolding strategies, which are laborious and sometime inefficient, are used to obtain correctly-folded active proteins. In the current study, we show that large quantities of correctly folded and completely active scFv molecules are there in bacterial inclusion bodies; they only need to be isolated from inclusion bodies...
October 31, 2018: Protein Expression and Purification
Shuai-Bing Zhang, Wei-Ji Zhang, Huan-Chen Zhai, Yang-Yong Lv, Jing-Ping Cai, Feng Jia, Jin-Shui Wang, Yuan-Sen Hu
β-1,3-glucanases, the plant PR-2 family of pathogenesis-related (PR) proteins, can be constitutively expressed and induced in wheat crop to enhance its anti-fungal pathogen defense. This study aimed to investigate the inhibitory effect of wheat β-1,3-glucanase on fungi most commonly associated with wheat kernel. A β-1,3-glucanase from wheat was successfully expressed in Pichia pastoris X-33 and its biochemical and antifungal properties were characterized herein. The molecular weight of recombinant β-1,3-glucanase is approximately 33 kDa...
October 28, 2018: Protein Expression and Purification
Nuo Xu, Bao Hui Wang, Qianyun Zhou, Yuehong Ouyang, Weiyue Gong, Haishan Tian, Xiaokun Li, Chao Jiang
Fibroblast growth factor 18 (FGF18) is a member of the fibroblast growth factor family and important in cartilage growth and development. However, the mechanism by which FGF18 mediates its biological functions is still unclear. In our study, we expressed the rhFGF18 protein fused to a HaloTag, (Halo-rhFGF18). MTT assay results indicated that both rhFGF18 and Halo-rhFGF18 have similar biological activities in NIH3T3 cells. However, basic FGF and acidic FGF were more potent than both rhFGF18 and Halo-rhFGF18...
October 26, 2018: Protein Expression and Purification
Lendsey Thicklin, Abdullah Shamsuddin, Fiezah Alahmry, Claire Gezley, Erika Brown, James Stone, Elizabeth Burns-Carver, Paul C Kline
A non-specific nucleoside hydrolase has been isolated from germinated Alaska pea seeds. The enzyme catalyzes the hydrolysis of both purines and pyrimidines along with ribo- and deoxyribonucleosides. A purification scheme utilized ammonium sulfate precipitation, ion exchange chromatography and size exclusion chromatography, resulted in 103-fold purification with a recovery of 2.8%. The purified protein has a specific activity of 0.308 μmol/min•mg. The subunit molecular weight was 26103 Da and the enzyme exists as a dimer...
October 24, 2018: Protein Expression and Purification
Nor Azura Azami, Ira Aryani Wirjon, Aik-Hong Teh, A A Amirul
Depolymerase is an enzyme that plays an important role in the hydrolysis of polyhydroxyalkanoates [PHAs]. In the current study, Burkholderia cepacia DP1 was obtained from Penang, Malaysia in which the enzyme was purified using ion exchange and gel filtration (Superdex-75) column chromatography. The molecular mass of the enzyme was estimated to be 53.3 kDa using SDS-PAGE. The enzyme activity was increased to 36.8 folds with the recovery of 16.3% after purification. The enzyme activity was detected between pH 6...
October 20, 2018: Protein Expression and Purification
Fatma Ersöz, Mehmet İnan
Milk-clotting enzymes used in the dairy industry can be obtained from different sources such as plants, animals, and microorganisms. Recombinant chymosin is the best alternative for the dairy industry due to the differences in physicochemical properties of coagulating enzymes and scarcity of chymosin from animal sources. In this study, glycosylated and non-glycosylated forms of yak chymosin were extracellularly produced in a methylotrophic yeast, Komagataella phaffii (Pichia pastoris). Synthetic yak prochymosin genes were cloned into the pPICZαA vector, expressed in P...
October 16, 2018: Protein Expression and Purification
Avijit Das, Malabika Biswas
The genome of aureophage Phi11 reveals the presence of the gene gp07 which codes for the putative antirepressor protein (GenBank accession no. NC_004615.1). Antirepressor proteins are mainly involved in lytic cycle determination mechanisms of various bacteriophages. The Phi11 protein Gp07 consists of two domains-an amino terminal Bro domain and a carboxy terminal KilA domain. Despite the important role of antirepressor proteins in the developmental pathway of phages, there are no reports on the purification and characterization of aureophage antirepressor proteins...
October 13, 2018: Protein Expression and Purification
Zhou Chen, Ahmed A Zaky, Yangliu Liu, Yaoyao Chen, Lu Liu, Siting Li, Yingmin Jia
A new extracellular xylanase was purified from a non-toxic mesophilic fungus Aspergillus flavus, and characterized as the β-1, 4-endoxylanase (designated as AfXynA) that appeared in a single protein band on SDS-PAGE with a molecular mass of 20.2 kDa, which is different from all other reported xylanases from the same strain. The AfXynA exhibited a specific activity of 838.2 U/mg. Its optimal temperature and pH were determined to be 55 °C and 7.5, respectively. It was stable up to 50 °C and within pH 3...
October 11, 2018: Protein Expression and Purification
Hongbo Li, Yuxian Xia
Short-chain insecticidal neurotoxin Tx4(6-1) from the spider Phoneutria nigriventer can be prepared by reversed-phase high-performance liquid-chromatography (HPLC) fractionation of PhTx4, but this is difficult and represents an obstacle preventing analyses of its insecticidal activity against agricultural insect pests. Herein, we performed secretory expression of recombinant Tx4(6-1) using Pichia pastoris strain X33 as the host, and screened transformants using enzyme-linked immunosorbent assay (ELISA). In flasks, ∼5 mg/l rTx4(6-1) was expressed as a secreted protein following induction with methanol, and this was increased to 45 mg/l rTx4(6-1) in a fed-batch reactor...
October 4, 2018: Protein Expression and Purification
V A Golotin, A P Filshtein, I V Chikalovets, Kim N Yu, V I Molchanova, O V Chernikov
The gene of mtl from the mussel Mytilus trossulus was cloned into pET-40b(+) expression vector. After expression in E. coli using designed MX-medium an instable soluble form of MTL was obtained. The developed isolation method of the recombinant protein in "semi-denatured" conditions allowed obtaining an active soluble form of the homogenous lectin from the mussel M. trossulus (r-MTL). Both of the lectins had similar antigenic and spatial structures.
October 4, 2018: Protein Expression and Purification
Chihoko Kobayashi, Masahiko Kato, Hidekazu Nagaya, Nobutaka Shimizu, Kazuhiro Ishibashi, Masayuki Ishikawa, Etsuko Katoh
Tomato mosaic virus (ToMV; genus, Tobamovirus) is a member of the alpha-like virus superfamily of positive-strand RNA viruses, which includes many plant and animal viruses of agronomical and clinical importance. The genomes of alpha-like viruses encode replication-associated proteins that contain methyltransferase, helicase and/or polymerase domains. The three-dimensional structure of the helicase domain fragment of ToMV has been determined, but the structures of the other domains of alpha-like virus replication proteins are not available...
October 3, 2018: Protein Expression and Purification
Wenxi Yu, Maiya Yu, Makaía M Papasergi-Scott, Gregory G Tall
Resistance to Inhibitors of Cholinesterase-8 (Ric-8) proteins are molecular chaperones that fold heterotrimeric G protein α subunits shortly after biosynthesis. Ric-8 proteins also act as test tube guanine nucleotide exchange factors (GEF) that promote Gα subunit GDP for GTP exchange. The GEF and chaperoning activities of Ric-8A are regulated by phosphorylation of five serine and threonine residues within protein kinase CK2 consensus sites. The traditional way that Ric-8A proteins have been purified is from Spodoptera frugiperda (Sf9) or Trichoplusia ni (Tni) insect cells...
October 2, 2018: Protein Expression and Purification
Ludmilla Dela Coletta Troiano Araújo, Márcia Wibrantz, Daniel Ernesto Rodríguez-Fernández, Susan Grace Karp, Alexandra Costa Talevi, Emanuel Maltempi de Souza, Carlos Ricardo Soccol, Vanete Thomaz-Soccol
The aim of this study was to evaluate the parameters that affect the production of the recombinant 10 kDa culture filtrate protein (CFP10), a promising reagent of high specificity for intradermoreaction and other antigen-based methods used in the diagnosis of tuberculosis. Conditions of Escherichia coli growth temperature, induction temperature and IPTG-inducer concentration were evaluated in shake flasks and dissolved O2 concentrations of 15 and 30% were evaluated in a bioreactor. The process parameters defined on small scale were: growth temperature between 30 and 37 °C, induction temperature of 26 °C and IPTG concentration of 0...
September 24, 2018: Protein Expression and Purification
Anis Farhan Fatimi Ab Wahab, Noor Adila Abdul Karim, Jonathan Guyang Ling, Nurain Shahera Hasan, Hui Yee Yong, Izwan Bharudin, Shazilah Kamaruddin, Farah Diba Abu Bakar, Abdul Munir Abdul Murad
Cellobiohydrolases catalyze the processive hydrolysis of cellulose into cellobiose. Here, a Trichoderma virens cDNA predicted to encode for cellobiohydrolase (cbhI) was cloned and expressed heterologously in Aspergillus niger. The cbhI gene has an open reading frame of 1518 bp, encoding for a putative protein of 505 amino acid residues with a calculated molecular mass of approximately 54 kDa. The predicted CbhI amino acid sequence has a fungal type carbohydrate binding module separated from a catalytic domain by a threonine rich linker region and showed high sequence homology with glycoside hydrolase family 7 proteins...
September 24, 2018: Protein Expression and Purification
Xiaojie Pei, Yaoguang Chang, Jingjing Shen
Alginate is the major constituent of brown algae and a commercially important polysaccharide with wide applications. Alginate lyases are desired tools for degrading alginate. Based on the genome mining of marine bacterium Wenyingzhuangia funcanilytica, an alginate lyase Aly7B_Wf was discovered, cloned and expressed in Escherichia coli. Aly7B_Wf belonged to subfamily 6 of PL7 family. Its biochemical properties, kinetic constants, substrate specificity and degradation pattern were clarified. The enzyme is an endo-acting bifunctional alginate lyase, and preferably cleaved polymannuronate (polyM)...
September 21, 2018: Protein Expression and Purification
Elizabeth Yihui Ng, Ying Ru Loh, Yan Li, Qingxin Li, CongBao Kang
The Zika virus (ZIKV) genome encodes a polyprotein that can be post-translationally processed into functional viral proteins. The viral protease is indispensable in the maturation of viral proteins. The Zika protease comprises of two components crucial for catalysis. The N-terminal region of NS3 contains the catalytic triad and approximately 40 amino acids of NS2B are essential for folding and protease activity. NS2B is a membrane protein with transmembrane domains that are critical for the localization of NS3 to the membrane...
September 21, 2018: Protein Expression and Purification
Ganfei Xu, Jingjing Wang, Qiang Yin, Wei Fang, Yazhong Xiao, Zemin Fang
The fungal laccase Lcc9 from Coprinopsis cinerea is a promising candidate for biotechnological applications due to its distinct biochemical properties. In the present work, Lcc9 cDNA was cloned from C. cinerea using reverse transcription polymerase chain reaction and heterologously expressed in Pichia pastoris GS115. The recombinant laccase was found to be a heavily hyperglycoprotein, with the molecular weight of 60.2 kDa as determined by MALDI-TOF. Laccase activity in the culture supernatant was 1750 ± 83 U/L and reached 3138 ± 62 U/L after expression condition optimization using orthogonal experiment...
September 21, 2018: Protein Expression and Purification
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