Secretory expression of bovine herpesvirus type 1/5 glycoprotein E in Pichia pastoris for the differential diagnosis of vaccinated or infected cattle
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. Following expression, the recombinant gE (rgE) was secreted and purified from the culture medium. The rgE was identified by Western blotting (WB) using sera from cattle naturally infected with BoHV-1 and/or BoHV-5, or sera from bovines experimentally infected with wild-type BoHV-5. Sera collected from cattle vaccinated with a BoHV-5 gI/gE/US9¯ marker vaccine failed to recognise rgE. Expression of rgE, based on a sequence containing highly conserved regions from BoHV-1 an...
Source: Protein Expression and Purification - October 10, 2016 Category: Biochemistry Source Type: research

Towards universal approach for bacterial production of three-finger Ly6/uPAR proteins: Case study of cytotoxin I from cobra N.  oxiana
Publication date: February 2017 Source:Protein Expression and Purification, Volume 130 Author(s): 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 ...
Source: Protein Expression and Purification - October 7, 2016 Category: Biochemistry Source Type: research

Biosynthesis, purification and characterization of β-1,4-xylanase from a novel mangrove associated actinobacterium Streptomyces olivaceus (MSU3) and its applications
Publication date: February 2017 Source:Protein Expression and Purification, Volume 130 Author(s): 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 i...
Source: Protein Expression and Purification - October 7, 2016 Category: Biochemistry Source Type: research

Oligo-clonal nanobodies as an innovative targeting agent for cancer therapy: New biology and novel targeting systems
In this study, we designed a robust prokaryotic expression system to express functional VHHs against HER2 receptor. This system showed high recombinant yields besides purified VHHs flow cytometry verified great capabilities of these molecules to pinpoint ecto-domain of HER2 receptor in MC4L2 HER2+ while insignificant non-specific binding to MC4L2 HER2-confirm nanobodies trivial cross-reaction. In the next step, we evaluated cooperative effect of four distinctive VHHs (oligoclonal VHHs) targeting different epitopes on HER2. As our result proved, using oligoclonal nanobodies as targeting moiety enhance targeting efficacy in ...
Source: Protein Expression and Purification - October 4, 2016 Category: Biochemistry Source Type: research

High-level expression of l-glutamate oxidase in Pichia pastoris using multi-copy expression strains and high cell density cultivation
Publication date: January 2017 Source:Protein Expression and Purification, Volume 129 Author(s): 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 re...
Source: Protein Expression and Purification - October 2, 2016 Category: Biochemistry Source Type: research

Purification, characterization and the use of recombinant prolyl oligopeptidase from Myxococcus xanthus for gluten hydrolysis
In this study recombinant Myxococcus xanthus prolyl oligopeptidase expressed in E. coli was purified 60.3 fold, using metal-chelate affinity and gel permeation chromatography. The recombinant enzyme had a monomeric molecular weight of 70 kDa. Isoelectric point of the enzyme was found to be approximately 6.3 by two-dimensional polyacrylamide gel electrophoresis. The optimum pH and temperature was estimated as 7.5 and 37 °C, respectively. The purified enzyme was stable in a pH range of 6.0–8.5 and thermally stable up to 37 °C. The Km and Vmax values were 0.2 mM and 3.42 μmol/min...
Source: Protein Expression and Purification - October 2, 2016 Category: Biochemistry Source Type: research

Evaluation of the virus clearance capacity and robustness of the manufacturing process for the recombinant factor VIII protein, turoctocog alfa
Publication date: January 2017 Source:Protein Expression and Purification, Volume 129 Author(s): Tracey W. Ellgaard, Lene Bindslev, Søren Kamstrup Turoctocog alfa is a B-domain-truncated recombinant factor VIII protein produced in a Chinese hamster ovary (CHO) cell line. The aim of this study was to evaluate the virus clearance capacity and robustness of the turoctocog alfa purification process. Virus clearance evaluation studies were conducted utilising a scaled-down version of the manufacturing process. Total virus clearance was evaluated using the ecotropic murine leukaemia virus (eMuLV) as a model for non-infec...
Source: Protein Expression and Purification - October 2, 2016 Category: Biochemistry Source Type: research

New findings in potential applications of tobacco osmotin
Publication date: January 2017 Source:Protein Expression and Purification, Volume 129 Author(s): Jitka Viktorova, Katerina Rehorova, Lucie Musilova, Jachym Suman, Petra Lovecka, Tomas Macek The osmotin protein is involved in both monocot and dicot plant responses to biotic and abiotic stress. To determine the biological activity of osmotin, the gene was amplified from tobacco genomic DNA, fused with the hexahistidine tag motif and successfully expressed in Escherichia coli, after which the recombinant osmotin was purified and renatured. Various activities were then tested, including hemolytic activity, toxicity against hu...
Source: Protein Expression and Purification - October 1, 2016 Category: Biochemistry Source Type: research

Recombinant production, purification and characterization of vessel dilator in E.  coli
Publication date: January 2017 Source:Protein Expression and Purification, Volume 129 Author(s): 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 H...
Source: Protein Expression and Purification - October 1, 2016 Category: Biochemistry Source Type: research

Efficient secretory expression of recombinant proteins in Escherichia coli with a novel actinomycete signal peptide
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. The thermophilic α-amylase gene of Bacillus licheniformis was fused with Kp-SP. Both recombinants, carrying inducible and constitutive plasmids, showed remarkable increases in extracellular and intracellular amylolytic activity. Amylase activity was observed to be...
Source: Protein Expression and Purification - October 1, 2016 Category: Biochemistry Source Type: research

Metabolic engineering of E.  coli top 10 for production of vanillin through FA catabolic pathway and bioprocess optimization using RSM
Publication date: December 2016 Source:Protein Expression and Purification, Volume 128 Author(s): Debkumar Chakraborty, Gaganjot Gupta, Baljinder Kaur Metabolic engineering and construction of recombinant Escherichia coli strains carrying feruloyl-CoA synthetase and enoyl-CoA hydratase genes for the bioconversion of ferulic acid to vanillin offers an alternative way to produce vanillin. Isolation and designing of fcs and ech genes was carried out using computer assisted protocol and the designed vanillin biosynthetic gene cassette was cloned in pCCIBAC expression vector for introduction in E. coli top 10. Recombinant...
Source: Protein Expression and Purification - September 24, 2016 Category: Biochemistry Source Type: research

Expression and purification of polioviral proteins in E.  coli, and production of antisera as reagents for immunological assays
Publication date: December 2016 Source:Protein Expression and Purification, Volume 128 Author(s): Madala Uma, P.P. Rao, K. Nagalekshmi, N.R. Hegde Poliomyelitis, caused by poliovirus, is on the verge of eradication, and the world is preparing to shift from live to inactivated polio vaccine. In view of the requirement of non-infectious reagents, especially protein antigens, for surveillance during the final phase of poliovirus eradication, we have attempted to generate reagents that may be of use for the development of diagnostic tests. Polioviral proteins VP0, VP3, VP1, and 3AB were expressed in Escherichia coli using the...
Source: Protein Expression and Purification - September 24, 2016 Category: Biochemistry Source Type: research

Identification and characterization of a novel amidase signature family amidase from Parvibaculum lavamentivorans ZJB14001
Publication date: January 2017 Source:Protein Expression and Purification, Volume 129 Author(s): Zhe-Ming Wu, Ren-Chao Zheng, Yu-Guo Zheng Amidase signature (AS) family amidases are known to exhibit broad substrate specificity. According to the available genome sequence data, a novel AS family amidase, Pl-Ami, was identified and cloned from the genome of Parvibaculum lavamentivorans ZJB14001. The recombinant amidase was overexpressed in Escherichia coli BL21, purified and functionally characterized. The optimal pH and temperature for Pl-Ami were 9.5 and 45 °C, respectively. Pl-Ami preferred long chain aliphatic a...
Source: Protein Expression and Purification - September 24, 2016 Category: Biochemistry Source Type: research

Production of recombinant dengue non-structural 1 (NS1) proteins from clinical virus isolates
This study was aimed to generate dengue NS1 recombinant protein for the development of dengue diagnostic tests. Four Indonesian DENV isolates were used as the source of the NS1 gene cloning, expression, and purification in bacterial expression system. Recombinant NS1 proteins were successfully purified and their antigenicities were assessed. Immunization of mice with recombinant proteins observed the immunogenicity of the NS1 protein. The generated recombinant proteins can be potentially used in the development of NS1 diagnostic test. With minimal modifications, this method can be used for producing NS1 recombinant protein...
Source: Protein Expression and Purification - September 24, 2016 Category: Biochemistry Source Type: research

A study of the small-molecule system used to investigate the effect of arginine on antibody elution in hydrophobic charge-induction chromatography
Publication date: January 2017 Source:Protein Expression and Purification, Volume 129 Author(s): Atsushi Hirano, Takuya Maruyama, Kentaro Shiraki, Tsutomu Arakawa, Tomoshi Kameda Hydrophobic charge-induction chromatography (HCIC) using 4-mercaptoethylpyridine (4-MEP) as the ligand is used to purify antibodies. The 4-MEP resin ligand has high affinity for antibodies, which makes it difficult to optimize the elution conditions. Recent studies showed that arginine is effective at eluting and purifying antibodies using the HCIC with 4-MEP. In the present study, we investigated the mechanism of the action of arginine on the in...
Source: Protein Expression and Purification - September 24, 2016 Category: Biochemistry Source Type: research

Biophysical and functional characterization of the human olfactory receptor OR1A1 expressed in a mammalian inducible cell line
Publication date: January 2017 Source:Protein Expression and Purification, Volume 129 Author(s): Christine Belloir, Marie-Louise Miller-Leseigneur, Fabrice Neiers, Loïc Briand, Anne-Marie Le Bon Olfactory receptors (ORs) play a crucial role in detecting the odorant molecules present in the surrounding environment. These receptors, which belong to class A G-protein-coupled receptors, constitute the largest transmembrane protein family in the human genome. Functional studies showed that the OR family includes members that are able to respond to a large set of odorants and members that are activated by a relatively smal...
Source: Protein Expression and Purification - September 20, 2016 Category: Biochemistry Source Type: research

Partitioning of xylanase from Thermomyces lanuginosus in PEG/NaCit aqueous two-phase systems: Structural and functional approach
Publication date: January 2017 Source:Protein Expression and Purification, Volume 129 Author(s): Dana B. Loureiro, Mauricio Braia, Diana Romanini, Gisela Tubio The structure and catalytic activity of xylanase from Thermomyces lanuginosus were studied in different media (containing polyethylene glycol -PEG- or salt) at different temperatures. The aim was to study how the native structure of the enzyme is affected to understand the partitioning behavior of xylanase in PEG/sodium citrate (PEG/NaCit) aqueous two-phase systems. The presence of PEGs of different molar masses slightly altered the native structure of xylanase, al...
Source: Protein Expression and Purification - September 20, 2016 Category: Biochemistry Source Type: research

Going native: Complete removal of protein purification affinity tags by simple modification of existing tags and proteases
Publication date: January 2017 Source:Protein Expression and Purification, Volume 129 Author(s): Hui Chin Goh, Radoslaw M. Sobota, Farid J. Ghadessy, Saurabh Nirantar Protein purification typically involves expressing a recombinant gene comprising a target protein fused to a suitable affinity tag. After purification, it is often desirable to remove the affinity tag to prevent interference with downstream functions of the target protein. This is mainly accomplished by placing a protease site between the tag and the target protein. Typically, a small oligopeptide ‘stub’ C-terminal to the cleavage site remains at...
Source: Protein Expression and Purification - September 18, 2016 Category: Biochemistry Source Type: research

Purification, expression and characterization of a novel α-l-fucosidase from a marine bacteria Wenyingzhuangia fucanilytica
This study successfully purified, identified, cloned, expressed and characterized a novel α-l-fucosidase, and meanwhile revealed a new multidomain structure of glycoside hydrolase. The knowledge gained from this study should facilitate the further research and application of α-l-fucosidases. (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - September 18, 2016 Category: Biochemistry Source Type: research

Strategies for over-expression and purification of recombinant full length STAT5B in Escherichia coli
Publication date: January 2017 Source:Protein Expression and Purification, Volume 129 Author(s): Elvin D. de Araujo, Mulu Geletu, Patrick T. Gunning STAT5B, a ubiquitious transcription factor, has been implicated in the onset and progression of several cancers. Since the inhibition of STAT activity holds significant therapeutic potential, there is a need to develop high-throughput biophysical screening platforms to rapidly identify high affinity binders of STATs. Biophysical assays would benefit from the efficient and cost-effective production of high purity, full-length STAT proteins. Herein, we have sampled a large regi...
Source: Protein Expression and Purification - September 18, 2016 Category: Biochemistry Source Type: research

Expression, purification and characterization of a full-length recombinant HIV-1 Vpu from inclusion bodies
Publication date: December 2016 Source:Protein Expression and Purification, Volume 128 Author(s): Zikhona Njengele, Ronel Kleynhans, Yasien Sayed, Salerwe Mosebi Vpu is one of four accessory proteins encoded by human immunodeficiency virus type I (HIV-1). Vpu modulates the expression of several cellular restriction factors within the HIV-1 infected cell including CD4, CD74, the bone marrow stromal antigen 2 (BST-2) and NK-T-and-B antigen. The interaction of HIV-1 Vpu with these proteins interferes with the innate immune response directed against HIV-1; thereby promoting viral persistence. The involvement of HIV-1 Vpu in m...
Source: Protein Expression and Purification - September 9, 2016 Category: Biochemistry Source Type: research

CRISPR-Cas9 mediated genetic engineering for the purification of the endogenous integrator complex from mammalian cells
Publication date: December 2016 Source:Protein Expression and Purification, Volume 128 Author(s): David Baillat, William K. Russell, Eric J. Wagner The Integrator Complex (INT) is a large multi-subunit protein complex, containing at least 14 subunits and a host of associated factors. These protein components have been established through pulldowns of overexpressed epitope tagged subunits or by using antibodies raised against specific subunits. Here, we utilize CRISPR/Cas9 gene editing technology to introduce N-terminal FLAG epitope tags into the endogenous genes that encode Integrator subunit 4 and 11 within HEK293T cells...
Source: Protein Expression and Purification - September 2, 2016 Category: Biochemistry Source Type: research

Protein expression of preferred human codon-optimized Gaussia luciferase genes with an artificial open-reading frame in mammalian and bacterial cells
Publication date: December 2016 Source:Protein Expression and Purification, Volume 128 Author(s): Satoshi Inouye, Takahiro Suzuki The protein expressions of three preferred human codon-optimized Gaussia luciferase genes (pGLuc, EpGLuc, and KpGLuc) were characterized in mammalian and bacterial cells by comparing them with those of wild-type Gaussia luciferase gene (wGLuc) and human codon-optimized Gaussia luciferase gene (hGLuc). Two synthetic genes of EpGLuc and KpGLuc containing the complete preferred human codons have an artificial open-reading frame; however, they had the similar protein expression levels to those of p...
Source: Protein Expression and Purification - August 29, 2016 Category: Biochemistry Source Type: research

A simple method for recombinant protein purification using self-assembling peptide-tagged tobacco etch virus protease
Publication date: December 2016 Source:Protein Expression and Purification, Volume 128 Author(s): Guang-Ya Li, Zhen-Zhen Xiao, Hui-Peng Lu, Yang-Yang Li, Xiao-Hui Zhou, Xiao Tan, Xin-Yu Zhang, Xiao-Li Xia, Huai-Chang Sun Recombinant protein purification remains to be a major challenge in biotechnology and medicine. In this paper we report a simple method for recombinant protein purification using self-assembling peptide-tagged tobacco etch virus protease (TEVp). After construction of an N-terminal ELK16 peptide fusion expression vector, we expressed ELK16-TEVp fusion protein in E. coli. SDS-PAGE analysis showed that ...
Source: Protein Expression and Purification - August 28, 2016 Category: Biochemistry Source Type: research

Expression, purification and enzymatic characterization of Brugia malayi dihydrofolate reductase
Publication date: December 2016 Source:Protein Expression and Purification, Volume 128 Author(s): Romy Perez-Abraham, Karla Garabiles Sanchez, Melany Alfonso, Ueli Gubler, John J. Siekierka, Nina M. Goodey Brugia malayi (B. malayi) is one of the three causative agents of lymphatic filariasis, a neglected parasitic disease. Current literature suggests that dihydrofolate reductase is a potential drug target for the elimination of B. malayi. Here we report the recombinant expression and purification of a ∼20 kDa B. malayi dihydrofolate reductase (BmDHFR). A His6-tagged construct was expressed in ...
Source: Protein Expression and Purification - August 26, 2016 Category: Biochemistry Source Type: research

Expression of recombinant Newcastle disease virus F protein in Pichia pastoris and its immunogenicity using flagellin as the adjuvant
In this study, we focus on F protein, the neutralizing and protective antigen of NDV, and flagellin (FliC), a toll-like receptor 5 (TLR5) agonist that is an effective inducer of innate immune responses. We amplified F gene from velogenic NDV strain F48E8. The recombinant histidine (His)-tagged F protein was efficiently expressed in a Pichia pastoris (P. pastoris) eukaryotic system and verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. The conditions for F protein expression in P. pastoris were optimal. The immunogenicity of F protein with FliC as the adjuvant was evaluated...
Source: Protein Expression and Purification - August 26, 2016 Category: Biochemistry Source Type: research

Expression and purification of the kinase domain of PINK1 in Pichia pastoris
Publication date: December 2016 Source:Protein Expression and Purification, Volume 128 Author(s): Daichao Wu, Lingzhi Qu, Yang Fu, Jun Li, Longying Jiang, Xiaojuan Chen, Ming Guo, Zhuchu Chen, Lin Chen, Yongheng Chen PTEN-induced putative kinase 1 (PINK1) is a Ser/Thr kinase that specifically localizes on the mitochondrial membrane. It cooperates with Parkin to regulate mitochondrial quality control. Mutations in PINK1 protein which account for 8–15% of Parkinson's disease (PD), are the second most common cause of early-onset Autosomal Recessive Parkinson's disease (AR-PD). The lack of methods for PINK1 heterologous...
Source: Protein Expression and Purification - August 26, 2016 Category: Biochemistry Source Type: research

Development of CYB5-fusion monitoring system for efficient periplasmic expression of multimeric proteins in Escherichia coli
Publication date: December 2016 Source:Protein Expression and Purification, Volume 128 Author(s): Dmitri Dormeshkin, Andrei Gilep, Gennady Sergeev, Sergey Usanov Despite all advances of heterologous expression of recombinant proteins in Escherichia coli, expression of multidomain disulphide-rich proteins faces some problems due to the absence of the possibility to monitor the process in real-time. Here we provide a CYB5-fusion system – cytochrome b 5 fusion system for periplasmic expression of multimeric proteins with the possibility of process monitoring. We validated this system by Fab and scFv antibody fragments ...
Source: Protein Expression and Purification - August 26, 2016 Category: Biochemistry Source Type: research

Expression, purification and identification of a thermolysin-like protease, neutral protease I, from Aspergillus oryzae with the Pichia pastoris expression system
This study was designed to obtain high expression levels of neutral protease I from Aspergillus oryzae 3.042 by using Pichia pastoris GS115 as the host strain for industrial purposes. The coding sequence of the target gene was modified, synthesized, and then cloned into the expression vector pHBM905BDM, which harbored the d1+2 × 201 AOX1 promoter and the MF4I leader sequence. The recombinant plasmid was transformed into Pichia pastoris GS115. The recombinant strain was used for high-density fermentation in a 4-L fermenter. The yield of the target protein reached 12.87 mg/mL, and the enzyme activity was...
Source: Protein Expression and Purification - August 21, 2016 Category: Biochemistry Source Type: research

Expression, purification and characterization of Esx-1 secretion-associated protein EspL from Mycobacterium tuberculosis
In this study, we tried several fusion tags and various expression conditions to recombinantly express MtEspL. Through a four-step purification procedure, ultimately, we successfully prepared the full-length MtEspL in Escherichia coli BL21 (DE3) with a purity of 98%. The yields of the purified MtEspL protein were 14 mg/L in Luria Bertani medium and 5.6 mg/L in M9 minimal medium, respectively. Biophysical experiments showed that MtEspL existed in a dimeric form. Moreover, the 1H-15N HSQC spectrum recorded on MtEspL illustrates a favorable dispersion of the resonance peaks, indicating that the symmetric dimeric MtE...
Source: Protein Expression and Purification - August 13, 2016 Category: Biochemistry Source Type: research

Expression, purification and characterization of GAPDH-ChSase ABC I from Proteus vulgaris in Escherichia coli
In this study, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is stably expressed in different cells like normal cells and cancer cells and the expression is relatively insensitive to experimental conditions, was expressed as a fusion protein with ChSase ABC I. Results showed that the expression level and enzyme activity of GAPDH-ChSase ABC I were about 2.2 and 3.0 times higher than those of ChSase ABC I. By optimization of fermentation conditions, higher productivity of ChSase ABC I was achieved as 880 ± 61 IU/g wet cell weight compared with the reported ones. The optimal temperature and pH ...
Source: Protein Expression and Purification - August 11, 2016 Category: Biochemistry Source Type: research

Development of an automated mid-scale parallel protein purification system for antibody purification and affinity chromatography
Publication date: December 2016 Source:Protein Expression and Purification, Volume 128 Author(s): Chi Zhang, Alexander M. Long, Brooke Swalm, Ken Charest, Yan Wang, Jiali Hu, Craig Schulz, Wolfgang Goetzinger, Brian E. Hall Protein purification is often a bottleneck during protein generation for large molecule drug discovery. Therapeutic antibody campaigns typically require the purification of hundreds of monoclonal antibodies (mAbs) during the hybridoma process and lead optimization. With the increase in high-throughput cloning, faster DNA sequencing, and the use of parallel protein expression systems, a need for high-th...
Source: Protein Expression and Purification - August 11, 2016 Category: Biochemistry Source Type: research

High-yield fermentation and a novel heat-precipitation purification method for hydrophobin HGFI from Grifola frondosa in Pichia pastoris
Publication date: December 2016 Source:Protein Expression and Purification, Volume 128 Author(s): Dongmin Song, Zhendong Gao, Liqiang Zhao, Xiangxiang Wang, Haijin Xu, Yanling Bai, Xiuming Zhang, Markus B. Linder, Hui Feng, Mingqiang Qiao Hydrophobins are proteins produced by filamentous fungi with high natural-surfactant activities and that can self-assemble in interfaces of air–water or solid–water to form amphiphilic membranes. Here, we reported a high-yield fermentation method for hydrophobin HGFI from Grifola frondosa in Pichia pastoris, attaining production of 300 mg/L by keeping the dissolved oxyge...
Source: Protein Expression and Purification - August 10, 2016 Category: Biochemistry Source Type: research

Molecular cloning, expression, and functional analysis of the copper amine oxidase gene in the endophytic fungus Shiraia sp. Slf14 from Huperzia serrata
In this study, we cloned and expressed an SsCAO gene from a HupA-producing endophytic fungus, Shiraia sp. Slf14. Analysis of the deduced protein amino acid sequence showed that it contained the Asp catalytic base, conserved motif Asn-Tyr-Asp/Glu, and three copper-binding histidines. The cDNA of SsCAO was amplified and expressed in Escherichia coli BL21(DE3), from which a 76 kDa protein was obtained. The activity of this enzyme was tested, which provided more information about the SsCAO gene in the endophytic fungus. Gas Chromatograph-Mass Spectrometry (GC-MS) revealed that this SsCAO could accept cadaverine as a subst...
Source: Protein Expression and Purification - August 9, 2016 Category: Biochemistry Source Type: research

Cloning, expression and characterization of potential immunogenic recombinant hemagglutinin-neuraminidase protein of Porcine rubulavirus
In this study, we expressed the HN-PorPV-recombinant (rHN-PorPV) protein in an Escherichia coli system and analyzed the immune responses in mice. The HN gene was cloned from the reference strain PorPV-La Piedad Michoacan Virus (GenBank accession number BK005918), into the pDual expression vector. The expressed protein was identified at a molecular weight of 61.7 kDa. Three-dimensional modeling showed that the main conformational and functional domains of the rHN-PorPV protein were preserved. The antigenicity of the expressed protein was confirmed by Western blot with a monoclonal antibody recognizing the HN, and by te...
Source: Protein Expression and Purification - August 9, 2016 Category: Biochemistry Source Type: research

Expression, purification, enzymatic characterization and crystallization of glyceraldehyde-3-phosphate dehydrogenase from Naegleria gruberi, the first one from phylum Percolozoa
Publication date: November 2016 Source:Protein Expression and Purification, Volume 127 Author(s): Agnes Thiane Pereira Machado, Marcio Silva, Jorge Iulek Naegleria gruberi had its genome sequenced by Fritz-Laylin and collaborators in 2010. It is not pathogenic, but has characteristics similar to those of Naegleria fowleri, opportunistic pathogen that can cause fatal encephalitis in humans. N. gruberi genome has contributed to a better understanding of the primitive eukaryotic metabolism and revealed the complexity of several metabolic pathways. In this paper we describe the expression, purification, enzyme characteri...
Source: Protein Expression and Purification - August 4, 2016 Category: Biochemistry Source Type: research

Engineering, expression and purification of a chimeric fibrin-specific streptokinase
Publication date: Available online 3 August 2016 Source:Protein Expression and Purification Author(s): Mohammad Naser Taheri, Abbas Behzad-Behbahani, Gholamreza Rafiei Dehbidi, Saeede Salehi, Sedigheh Sharifzadeh Streptokinase is a valuable fibrinolytic agent used to cope with myocardial infarction and brain stroke. Despite its high efficiency in dissolving blood clots, streptokinase (SK) has no specificity in binding fibrin, causing some problems such as internal bleedings following its administration. To make streptokinase fibrin specific and limit the fibrinolytic process to the clot location, we engineered a chimeric ...
Source: Protein Expression and Purification - August 3, 2016 Category: Biochemistry Source Type: research

A simple and efficient method for generating high-quality recombinant Mical enzyme for in  vitro assays
Publication date: November 2016 Source:Protein Expression and Purification, Volume 127 Author(s): Heng Wu, Ruei-Jiun Hung, Jonathan R. Terman We have recently identified a new family of multidomain oxidoreductase (redox) enzymes, the MICALs, that directly regulate the actin cytoskeletal elements necessary for the morphology, motility, and trajectory of cells. Our genetic assays reveal that Mical is both necessary and sufficient for actin organization and cellular effects in vivo and our biochemical assays with purified Mical protein reveal that Mical utilizes its redox activity to directly disassemble actin filam...
Source: Protein Expression and Purification - July 29, 2016 Category: Biochemistry Source Type: research

BAY 81-8973, a full-length recombinant factor VIII: Human heat shock protein 70 improves the manufacturing process without affecting clinical safety
In conclusion, addition of HSP70 to the BAY 81-8973 cell line is an innovative technology for manufacturing rFVIII aimed at improving protein folding and expression. Improved pharmacokinetics and no effect on safety of BAY 81-8973 were observed in clinical trials in patients with hemophilia A. (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - July 26, 2016 Category: Biochemistry Source Type: research

Capto MMC mixed-mode chromatography of murine and rabbit antibodies
Publication date: November 2016 Source:Protein Expression and Purification, Volume 127 Author(s): Tsutomu Arakawa, Yasunori Kurosawa, Michael Storms, Toshiaki Maruyama, C.J. Okumura, Yoshiko Kita Murine antibodies have weak affinity for Protein-A. Here, we have tested binding of murine monoclonal antibody (mAb) to Protein-A or Protein-A/Protein-G mixture under salting-out conditions. The addition of ammonium sulfate to HEK conditioned medium (CM) expressing murine mAb resulted in complete binding, leading to its elution by low pH or neutral arginine solution. Alternatively, a mixed-mode chromatography using Capto M...
Source: Protein Expression and Purification - July 26, 2016 Category: Biochemistry Source Type: research

Strategies for increasing heterologous expression of a thermostable esterase from Archaeoglobus fulgidus in Escherichia coli
In this study, we used various strategies, such as changing the expression vector and host strain, codon optimization, and optimization of induction conditions, to increase the expression of Est-AF. Through codon optimization and by changing the vector and host strain, Est-AF expression was increased from 31.50 ± 0.35 mg/L to 61.75 ± 0.28 mg/L. The optimized expression system consisted of a codon-optimized Est-AF gene in a pET28a(+)-based expression plasmid in E. coli Rosetta cells. The expression level was further increased by optimizing the induction conditions. The optimi...
Source: Protein Expression and Purification - July 26, 2016 Category: Biochemistry Source Type: research

Identification and characterization of an immunogenic antigen, enolase 2, among excretory/secretory antigens (ESA) of Toxoplasma gondii
In conclusion, our results clearly show that the enzymatic activity of T. gondii enolase 2 is ion dependent and that it could be influenced by environmental factors. We also provide evidence that enolase 2 is an important immunogenic protein of ESAs from T. gondii and that it is a surface-exposed protein with strong antigenicity and immunogenicity. Our findings indicate that enolase 2 could play important roles in metabolism, immunogenicity and pathogenicity and that it may serve as a novel drug target and candidate vaccine against T. gondii infection. (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - July 26, 2016 Category: Biochemistry Source Type: research

Enhancement of a high efficient autoinducible expression system in Bacillus subtilis by promoter engineering
Publication date: November 2016 Source:Protein Expression and Purification, Volume 127 Author(s): Jintao Cheng, Chengran Guan, Wenjing Cui, Li Zhou, Zhongmei Liu, Weijiang Li, Zhemin Zhou Quorum-sensing related promoter srfA (PsrfA) was used to construct autoinducible expression system for production of recombinant proteins in Bacillus subtilis. PsrfA was prominent in the unique property of inducer-free activity that is closely correlated with cell density. Here, using green fluorescent protein (GFP) as the reporter protein, PsrfA was optimized by shortening its sequences and changing the nucleotides at the conser...
Source: Protein Expression and Purification - July 23, 2016 Category: Biochemistry Source Type: research

BacMam production of active recombinant lecithin –cholesterol acyltransferase: Expression, purification and characterization
Publication date: September 2016 Source:Protein Expression and Purification, Volume 125 Author(s): William G. Romanow, Derek E. Piper, Preston Fordstrom, Stephen Thibault, Mingyue Zhou, Nigel P.C. Walker Lecithin–cholesterol acyltransferase (LCAT) is a key enzyme in the esterification of cholesterol and its subsequent incorporation into the core of high density lipoprotein (HDL) particles. It is also involved in reverse cholesterol transport (RCT), the mechanism by which cholesterol is removed from peripheral cells and transported to the liver for excretion. These processes are involved in the development of ...
Source: Protein Expression and Purification - July 20, 2016 Category: Biochemistry Source Type: research

Expression, purification, refolding and in  vitro recovery of active full length recombinant human gelatinase MMP-9 in Escherichia coli
Publication date: October 2016 Source:Protein Expression and Purification, Volume 126 Author(s): Sara Mohseni, Tahereh Tohidi Moghadam, Bahareh Dabirmanesh, Khosro Khajeh Human gelatinase (MMP-9) is a member of matrix metalloproteinases family (MMPs), which has been associated with malignant tumor progression and metastasis by matrix degradation. Herein, active full length recombinant human MMP-9 (amino acid residues 107–707) has been expressed in the form of inclusion bodies in Escherichia coli BL21, using pET21a vector. Solubilization of inclusion bodies was carried out in Tris-HCl buffer with 6 M urea, ...
Source: Protein Expression and Purification - July 20, 2016 Category: Biochemistry Source Type: research

Purification, characterization, and crystallization of membrane bound Escherichia coli tyrosine kinase
In this study, an efficient protocol to produce full length Etk solubilized in n-dodecyl-β-d-maltoside has been established with high yield. We have determined that detergent solubilized Etk retains kinase activity, but the protein is prone to aggregation, degradation, and has been unsuccessful in protein crystallization trials. In response, we designed and characterized truncations of Etk that do not aggregate and have led to successful crystallization experiments. In this article, we discuss our optimized expression and purification protocol for Etk, the design of Etk protein truncations, and the behavior of Etk dur...
Source: Protein Expression and Purification - July 14, 2016 Category: Biochemistry Source Type: research

BacMam production of active recombinant lecithin–cholesterol acyltransferase: Expression, purification and characterization
Publication date: September 2016 Source:Protein Expression and Purification, Volume 125 Author(s): William G. Romanow, Derek E. Piper, Preston Fordstrom, Stephen Thibault, Mingyue Zhou, Nigel P.C. Walker Lecithin–cholesterol acyltransferase (LCAT) is a key enzyme in the esterification of cholesterol and its subsequent incorporation into the core of high density lipoprotein (HDL) particles. It is also involved in reverse cholesterol transport (RCT), the mechanism by which cholesterol is removed from peripheral cells and transported to the liver for excretion. These processes are involved in the development of ...
Source: Protein Expression and Purification - July 14, 2016 Category: Biochemistry Source Type: research

Transformation of Escherichia coli and protein expression using lipoplex mimicry
In this study, we provide a simple method for E. coli transformation, which does not require the preparation of competent cells. The present method also shortens the overall procedures for transformation and gene expression in E. coli by omitting the colony-forming step. (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - July 13, 2016 Category: Biochemistry Source Type: research

Expression of soluble human Neonatal Fc-receptor (FcRn) in Escherichia coli through modification of growth environment
In this study, we demonstrate a novel strategy to express the α-chain of FcRn using E. coli as the expression host. The expression vector that carries the cDNA of the α-chain was transformed into expression host, Rosetta-gami 2 strain for inducible expression. The bacterial culture was grown in a modified growth medium which constitutes of terrific broth, sodium chloride (NaCl), glucose and betaine. A brief heat shock at 45 °C was carried out after induction, before the temperature for expression was reduced to 22 °C and grown for 16 h. The soluble form of the α-chain of FcRn expres...
Source: Protein Expression and Purification - July 11, 2016 Category: Biochemistry Source Type: research

Recombinant human Tat-Hsp70-2: A tool for neuroprotection
Publication date: Available online 9 July 2016 Source:Protein Expression and Purification Author(s): Pamela Cappelletti, Elisa Binda, Marta Tunesi, Diego Albani, Carmen Giordano, Gianluca Molla, Loredano Pollegioni Human Hsp70-2 is a chaperone expressed mainly in the nervous system. Up to now, no study has reported on the recombinant expression of this important human chaperone. Herein, we describe the successful purification and characterization of recombinant human Hsp70-2 in Escherichia coli in both the full-length and the chimeric protein containing the protein transduction domain corresponding to the trans-ac...
Source: Protein Expression and Purification - July 10, 2016 Category: Biochemistry Source Type: research