Enzymatic characteristics of a recombinant protease (rPepD) from Aspergillus niger expressed in Pichia pastoris
Publication date: October 2019Source: Protein Expression and Purification, Volume 162Author(s): Ye Ke, Meng-yao Wei, Yu-tong Fu, Yan-mei Zhu, Xuan-lin ZhanAbstractThe Aspergillus niger AS3.350 protease gene (pepD) was successfully cloned and expressed in Pichia pastoris KM71. The rPepD activity was 331.5 U/ml, and the optimum temperature and pH were 45 °C and 8–9 respectively. In addition, enzyme activity was significantly inhibited by PMSF, EDTA, Mg2+, Fe2+ and Zn2+ ions, and stimulated by Ca2+ which selectively bound to the T302 and D323 residues. Mutation in either or both of the residues inhibited rPepD exp...
Source: Protein Expression and Purification - June 15, 2019 Category: Biochemistry Source Type: research

Purification and characterization of a novel wild-type α-amylase from Antarctic sea ice bacterium Pseudoalteromonas sp. M175
Publication date: Available online 11 June 2019Source: Protein Expression and PurificationAuthor(s): Xiaofei Wang, Guangfeng Kan, Cuijuan Shi, Qiuju Xie, Yun Ju, Ruiqi Wang, Yongping Qiao, Xiulian RenAbstractA novel wild-type α-amylase named wtAmy175 from Pseudoalteromonas sp. M175 strain was purified through ammonium sulphate precipitation, DEAE cellulose, and Sephadex G-75 sequentially (25.83-fold, 7.67%-yield) for biochemical characterization. SDS-PAGE and zymographic activity staining of purified enzyme showed a single band with a predicted molecular mass of about 61 kDa. The optimum temperature and pH for enzy...
Source: Protein Expression and Purification - June 12, 2019 Category: Biochemistry Source Type: research

Expression, purification and characterization of diguanylate cyclase from Rhodococcus ruber
Publication date: Available online 11 June 2019Source: Protein Expression and PurificationAuthor(s): Sufang Kuang, Yuan Yuan, Zhonghao Wu, Ren PengAbstractDiguanylate cyclases (DGCs) were responsible for the synthesis of second messenger cyclic di-guanosine monophosphate (c-di-GMP), which were involved in various physiological activities of bacterial species. Here, a full-length DGC from Rhodococcus ruber SD3 fused with glutathione-S-transferase (GST) was expressed in E. coli and purified by glutathione agarose resin. The apparent molecular mass of one subunit of the purified diguanylate cyclase with GST tag (GST-DGC) was ...
Source: Protein Expression and Purification - June 12, 2019 Category: Biochemistry Source Type: research

Soluble expression of mature Rhizopus chinensis lipase in Escherichia coli and enhancement of its ester synthesis activity
Publication date: Available online 8 June 2019Source: Protein Expression and PurificationAuthor(s): Zhang Zhang, Dong Wang, Yan XuAbstractThe production of membrane-associated lipase from Rhizopus chinensis (RCL), which has a high ester synthesis activity and important potential applications, is difficult in heterologous expression system such as Escherichia coli and often leads to the formation of inclusion bodies. Here, we describe the soluble expression of mature RCL (mRCL) using maltose-binding protein (MBP) as a solubility-enhancing tag in the E. coli system. Although the MBP-mRCL fusion protein was soluble, mRCL was ...
Source: Protein Expression and Purification - June 9, 2019 Category: Biochemistry Source Type: research

Study on cloning, expression and hydrolysis characteristics of Aspergillus niger protease (pepD) gene
Publication date: Available online 8 June 2019Source: Protein Expression and PurificationAuthor(s): Ye Ke, Meng-yao Wei, Yu-tong Fu, Yan-mei Zhu, Xuan-lin ZhanAbstractThe Aspergillus niger AS3.350 protease gene (pepD) was successfully cloned and expressed in Pichia pastoris KM71. The rPepD activity was 331.5 U/ml, and the optimum temperature and pH were 45 °C and 8–9 respectively. In addition, enzyme activity was significantly inhibited by PMSF, EDTA, Mg2+, Fe2+ and Zn2+ ions, and stimulated by Ca2+ which selectively bound to the T302 and D323 residues. Mutation in either or both of the residues inhibited rPepD...
Source: Protein Expression and Purification - June 9, 2019 Category: Biochemistry Source Type: research

Promiscuity of host cell proteins in the purification of histidine tagged recombinant xylanase a by IMAC procedures: A case study with a Ni2+-tacn-based IMAC system
This study explores how different experimental conditions affect the HCP profiles generated during the immobilised metal ion affinity chromatographic (IMAC) purification with a Ni2+-1,4,7-triaza-cyclononane (tacn) Sepharose FF™ sorbent of the Bacillus halodurans N- and C-terminal His6-tagged xylanase A, expressed by Escherichia coli BL21(DE3) cells, and captured directly from cell lysates. Comparative studies were also carried out under identical loading, wash and elution conditions using nitrilotriacetic acid (NTA), also immobilised onto an agarose support and complexed with Ni2+ ions. High-resolution tandem mass sp...
Source: Protein Expression and Purification - June 5, 2019 Category: Biochemistry Source Type: research

Expression and purification of the recombinant full-length retinoblastoma protein and characterisation of its interaction with the oncoprotein HDM2
Publication date: Available online 1 June 2019Source: Protein Expression and PurificationAuthor(s): Adriana Rousset-Roman, Yolanda Rebolloso-Gómez, Vanesa Olivares-IllanaAbstractRetinoblastoma (Rb) was the first tumour suppressor factor described, and it is dysfunctional in several types of cancers. Structurally, Rb is a very large, multifunctional protein organized in different domains connected by intrinsically disordered regions. Due to the complex structure of Rb, biochemical manipulation is difficult. The Rb protein has been implicated in many different cellular processes, such as the cell cycle control, senesc...
Source: Protein Expression and Purification - June 2, 2019 Category: Biochemistry Source Type: research

Detection of IgG directed against a recombinant form of Epstein-Barr virus BALF0/1 protein in patients with nasopharyngeal carcinoma
This study describes the expression and purification of a truncated form of BALF0/1 (tBALF0) using a heterologous bacterial expression system. tBALF0 was further used as an antigen in an indirect Enzyme-linked Immunosorbent Assay (ELISA) that unraveled the presence of low titer IgGs to BALF0/1 during primary (10.0%) and past (13.3%) EBV infection. Conversely high-titer IgGs to BALF0/1 were detected in 33.3% of nasopharyngeal carcinoma (NPC) patients suggesting that BALF0/1 and/or humoral response against it may contribute to NPC pathogenesis. (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - May 30, 2019 Category: Biochemistry Source Type: research

Cloning and expression of pullulanase from Bacillus subtilis BK07 and PY22 in Pichia pastoris
In this study, pullulanase genes from a wild isolate B. subtilis BK07 and B. subtilis PY22 (mutant strain derived from B. subtilis 168) were transformed into P. pastoris KM71H. Extracellular recombinant protein production was achieved with methanol induction under the regulation of AOX1 promoter utilizing the Saccharomyces cerevisiae α-mating factor sequence for extracellular secretion. The molecular weight of the recombinant enzymes BK07pul and PY22pul were both approximately 90 kDa. Both enzymes showed highest activity at 40 °C, however PY22pul showed optimum activity at pH 6 whereas, BK07pul had highest ac...
Source: Protein Expression and Purification - May 27, 2019 Category: Biochemistry Source Type: research

Expression, purification and characterization of the plant Snf1-related protein kinase 1 from Escherichia coli
Publication date: Available online 21 May 2019Source: Protein Expression and PurificationAuthor(s): Rong-Qing ZhaoAbstractThe SnRK1 (SNF1 related protein kinase 1), a plant homologue of SNF1 (Sucrose non-fermenting 1)/AMPK (AMP-activated protein kinase), is an important metabolic sensor involving in catabolic and anabolic processes. SnRK1 is essential for plant metabolism regulation and response to environmental stresses. The plant SnRK1 consists of one catalytic (α1/α2 subunit) and two regulatory subunits (β1/β2/β3 and γ/βγ subunits), and functions as a heterotrimeric complex. H...
Source: Protein Expression and Purification - May 22, 2019 Category: Biochemistry Source Type: research

Expression and purification of domain III proteins from Dengue and Zika viruses
Publication date: Available online 18 May 2019Source: Protein Expression and PurificationAuthor(s): Josselin Corzo-Gómez, Julio García Cordero, Alfredo E. Montes Gómez, Karen Bernal Siria, Karime Namorado-Tónix, Benito Gutiérrez Castañeda, Leticia Cedillo-BarrónAbstractThe envelope (E) protein from Dengue and Zika viruses comprises three functional and structural domains (DI, DII, and DIII). Domain III induces most of the neutralizing antibodies and, as such, is considered as having the highest antigenic potential for the evaluation of population-level surveillance and for d...
Source: Protein Expression and Purification - May 19, 2019 Category: Biochemistry Source Type: research

Expression in Lactococcus lactis of a β-1,3-1,4-glucanase gene from Bacillus sp. SJ-10 isolated from fermented fish
In this study, a BG gene was transformed into the food-grade plasmid pNZ8149 and successfully expressed in Lactococcus lactis NZ3900 using the nisin-controlled gene expression system. To facilitate extracellular secretion, the signal peptide Usp45 was added during vector construction. A histidine tag was also added for affinity purification. BG was extracellularly secreted and was also present in the cells in soluble form. N-terminal amino acid residue analysis of secreted BG revealed that the Usp45 peptide was removed. The optimum temperature and pH for both intracellular and extracellular BG were 40 °C and 6, respe...
Source: Protein Expression and Purification - May 19, 2019 Category: Biochemistry Source Type: research

Cloning and purification of an anti-thrombotic, chimeric Staphylokinase in Pichia pastoris
Publication date: Available online 17 May 2019Source: Protein Expression and PurificationAuthor(s): Vandana, Satish Kantipudi, Neeraj Maheshwari, Sheetal Sharma, Girish SahniAbstractThere has been an increasing prevalence of cardiovascular diseases such as myocardial infarction and stroke in modern societies because of multiple lifestyle related issues like sedentariness and obesity, alcohol consumption and many more “life-style”factors. The FDA-approved thrombolytics such as Tissue Plasminogen Activator, Streptokinase etc. are used to lyse the clots in thrombotic disorders such as myocardial infarction, stroke...
Source: Protein Expression and Purification - May 18, 2019 Category: Biochemistry Source Type: research

Editorial Board
Publication date: September 2019Source: Protein Expression and Purification, Volume 161Author(s): (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - May 18, 2019 Category: Biochemistry Source Type: research

Expression and characterization of albumin fusion protein canine IFNγ-CSA in baculovirus-insect cell expression system
In this study, canine IFNγ was fused by a flexible linker with canine serum albumin to construct the fusion protein IFNγ-CSA for the purpose to design a long-acting canine IFNγ. The fusion protein was successfully expressed in baculovirus-infected Sf9 insect cells and was purified by salting-out and ion exchange chromatography. The IFNγ-CSA fusion possessed potent anti-viral assay against vesicular stomatitis virus in cultured cells. IFNγ-CSA was also stable at 37 °C up to 72 h compared with 8 h for IFNγ alone. In vivo pharmacokinetics demonstrated a significantly longer half-l...
Source: Protein Expression and Purification - May 16, 2019 Category: Biochemistry Source Type: research

Editorial Board
Publication date: August 2019Source: Protein Expression and Purification, Volume 160Author(s): (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - May 16, 2019 Category: Biochemistry Source Type: research

Expression of soluble, glycosylated and correctly folded dengue virus NS1 protein in Pichia pastoris
Publication date: Available online 8 May 2019Source: Protein Expression and PurificationAuthor(s): Diego Allonso, Iuri B. Pereira, Ada MB. Alves, Eleonora Kurtenbach, Ronaldo Mohana-BorgesAbstractThe dengue virus (DENV) non structural protein 1 (NS1) is a 46–55 kDa protein that exists as homodimer inside cells and as hexamer in the extracellular milieu. Several lines of evidence have demonstrated that the biochemical and structural properties of recombinant NS1 (rNS1) vary depending on the protein expression system used. Aiming to improve current tools for studying NS1 multiple roles, a recombinant tag-free NS1 pro...
Source: Protein Expression and Purification - May 10, 2019 Category: Biochemistry Source Type: research

Selective separation method of aggregates from IgG solution by aqueous two-phase system
Publication date: Available online 1 May 2019Source: Protein Expression and PurificationAuthor(s): Chika Shibata, Kazuki Iwashita, Kentaro ShirakiAbstractAggregation of immunoglobulin G (IgG) is a serious concern that results in immunogenicity in pharmaceutical applications. Removal of the small and soluble aggregates in protein solutions through a simple method remains challenging. Here we show that an aqueous two-phase system (ATPS) can be used for the elimination of soluble aggregates from IgG solution. Polyethylene glycol (PEG) and dextran (DEX) were selected as components of the ATPS. As expected, IgG monomers were pa...
Source: Protein Expression and Purification - May 2, 2019 Category: Biochemistry Source Type: research

Overproduction and purification of the Escherichia coli transcription factors “toxic” to a bacterial cell
Publication date: Available online 1 May 2019Source: Protein Expression and PurificationAuthor(s): Tatiana A. Bessonova, Natalia V. Lekontseva, Uliana S. Shvyreva, Alexey D. Nikulin, Maria N. Tutukina, Olga N. OzolineAbstractTranscription factors play a crucial role in control of life of a bacterial cell, working as switchers to a different life style or pathogenicity. To reconstruct the network of regulatory events taking place in changing growth conditions, we need to know regulons of as many transcription factors as possible, and motifs recognized by them. Experimentally this can be attained via ChIP-seq in vivo, SELEX ...
Source: Protein Expression and Purification - May 2, 2019 Category: Biochemistry Source Type: research

Preparation and identification of polyclonal antibody against human cytomegalovirus encoding protein UL23
Publication date: Available online 1 May 2019Source: Protein Expression and PurificationAuthor(s): Huizi Chai, Songbin Wu, Jinfeng Deng, Linyuan Feng, Xiaoping Yang, Yanhong Ran, Hongjian LiAbstractHuman cytomegalovirus (HCMV), a member of the human herpesvirus family, is a common opportunistic virus causing severe ailments and deaths in people with immature or compromised immune systems. UL23 is a virion protein found in the tegument and is expressed in the cytoplasm in HCMV infected cells. However, UL23 is dispensable for viral replication in cultured cells and little is currently known about its function. In order to fu...
Source: Protein Expression and Purification - May 2, 2019 Category: Biochemistry Source Type: research

Sequence optimization and glycosylation of vasoinhibin: Pitfalls of recombinant production
Publication date: Available online 30 April 2019Source: Protein Expression and PurificationAuthor(s): Bibiana Moreno-Carranza, Juan Pablo Robles, Hugo Cruces-Solís, Martha Gabriela Ferrer-Ríos, Eduardo Aguilar-Rivera, Marco Yupanki, Gonzalo Martínez de la Escalera, Carmen ClappAbstractVasoinhibin belongs to a family of proteins with antiangiogenic properties derived by proteolytic cleavage from the hormone prolactin (PRL). Vasoinhibin isoforms range from the first 79 to the first 159 residues of PRL. In an attempt to increase the yield of recombinant vasoinhibin and avoid incorrect intra- and inter-dis...
Source: Protein Expression and Purification - May 1, 2019 Category: Biochemistry Source Type: research

Expression, purification and spectrophotometric analysis of nucleoside hydrolase from Leishmania chagasi (LcNH)
We present here for the first time, the expression and purification protocols to obtain the enzymes LcNH1 and LcNH2 that can be employed to explore novel strategies to produce nucleoside hydrolase inhibitors for use in chemotherapy. Protein integrity was also confirmed by SDS-PAGE gel, mass spectrometry and enzymatic activity. (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - April 28, 2019 Category: Biochemistry Source Type: research

Expression, purification, and characterization of the Degludec precursor DesB30
In this study, a spacer peptide was added before the sequence of DesB30 and the C-peptide was replaced with AAK, a short connecting peptide. The target gene was cloned under the control of AOX1 and expressed as a secretory protein in Pichia pastoris. A high-yield recombination strain was selected by screening for resistance to G418. The basal salts medium was optimized and a lower salt concentration medium was found to show the best effects. Both media were used to compare the yield of high-density fermentation. The maximum yield reached 4.51 g/L in 1/2 basal salt medium, which is the highest reported yield to date. The ...
Source: Protein Expression and Purification - April 28, 2019 Category: Biochemistry Source Type: research

“CodonWizard” – An intuitive software tool with graphical user interface for customizable codon optimization in protein expression efforts
Publication date: August 2019Source: Protein Expression and Purification, Volume 160Author(s): Peter Rehbein, Jannik Berz, Patrick Kreisel, Harald SchwalbeAbstractOptimization of coding sequences to maximize protein expression yield is often outsourced to external service providers during commercial gene synthesis and thus unfortunately remains a black box for many researchers. The presented software program “CodonWizard” offers scientists a powerful but easy-to-use tool for customizable codon optimization: The intuitive graphical user interface empowers even scientists inexperienced in the art to straightforwa...
Source: Protein Expression and Purification - April 28, 2019 Category: Biochemistry Source Type: research

Expression, purification and biochemical characterization of Listeria monocytogenes single stranded DNA binding protein 1
Publication date: Available online 26 April 2019Source: Protein Expression and PurificationAuthor(s): Debika Ojha, M.V. Greeshma, K. Neelakanteshwar PatilAbstractSingle-stranded DNA binding proteins play an important role in DNA metabolic processes including replication, recombination, and repair. Here, we report the identification and biochemical characterization of the SSB1 protein from the foodborne pathogen Listeria monocytogenes. The L. monocytogenes SSB1 share 33% identity and 50.5% similarity with the prototype E. coli SSB protein. The electrophoretic mobility shift assay revealed that the purified L. monocytogenes ...
Source: Protein Expression and Purification - April 28, 2019 Category: Biochemistry Source Type: research

Biochemical and biophysical characterization of a prokaryotic Mg2+ ion channel: Implications for cost-effective purification of membrane proteins
Publication date: Available online 24 April 2019Source: Protein Expression and PurificationAuthor(s): Satyaki Chatterjee, Anindita Das, H. RaghuramanAbstractAlthough magnesium is the second most abundant cation present in the cell, the transport mechanism of Mg2+ across membranes is poorly understood. Importantly, the prokaryotic MgtE Mg2+ channel is related to mammalian SLC41A1 transporters and, therefore, biochemical and biophysical characterization of MgtE and its orthologs assumes significance. To date, the purification and structure determination of MgtE from Thermus thermophilus has been carried out using the widely ...
Source: Protein Expression and Purification - April 25, 2019 Category: Biochemistry Source Type: research

A versatile method for producing labeled or unlabeled Aβ55, Aβ40, and other β-amyloid family peptides
We present a straightforward, versatile method for expressing and purifying β-amyloid (Aβ40) and transmembrane peptides derived from β-amyloid precursor protein (Aβ55). In principle, these methods should be applicable to other types of strongly aggregating peptides. We start with a DNA plasmid encoding a HexaHis tag with a flexible, hydrophilic linker sequence, followed by a cleavage site, and then Aβ peptides. The HexaHis tag rather than a protein fusion partner (e.g., GST) obviates the need for a folded protein in affinity purification. Second, we present two cleavage methods, using either Factor...
Source: Protein Expression and Purification - April 23, 2019 Category: Biochemistry Source Type: research

A new vector coupling ligation-independent cloning with sortase a fusion for efficient cloning and one-step purification of tag-free recombinant proteins
Publication date: Available online 22 April 2019Source: Protein Expression and PurificationAuthor(s): Xinying Jia, Theo Crawford, Alan H. Zhang, Mehdi MobliAbstractWe have developed a new ligation independent cloning (LIC) vector – pSrtA9, which can be utilized for one-step purification of recombinant proteins. The new LIC site in the pSrtA9 vector, hosts a DNA sequence centered on a SfoI restriction site and integrates a coding sequence for sortase A (SrtA) recognition. Preceding the LIC site, pSrtA9 incorporates an N-terminal 6xHis-tag and the catalytic core of SrtA from Staphylococcus aureus (SrtAΔ59). Thus,...
Source: Protein Expression and Purification - April 23, 2019 Category: Biochemistry Source Type: research

Cloning, expression, and purification of the recombinant pro-apoptotic dominant-negative survivin T34A-C84A protein in Escherichia coli
In conclusion, our study provides a protocol of producing a biologically active survivin-targeting macromolecule, T34A-C84A-dNSur-His, which can be used as a tool for studying the molecular and cellular roles of survivin in cells. T34A-C84A-dNSur-His is also a potential therapeutic agent for augmenting cancer therapy. (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - April 19, 2019 Category: Biochemistry Source Type: research

Improvement of soluble expression of GM-CSF in the cytoplasm of Escherichia coli using chemical and molecular chaperones
Publication date: Available online 15 April 2019Source: Protein Expression and PurificationAuthor(s): Raziyeh Malekian, Setareh Sima, Ali Jahanian-Najafabadi, Fatemeh Moazen, Vajihe AkbariAbstractThe most common approaches to improve soluble expression of heterologous proteins are applications of molecular chaperones such as DnaK, DnaJ, GrpE, GroEL and GroES. The aim of present study was to enhance soluble expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in Escherichia coli by different approaches including modification of cultivation and induction conditions, and thermally, genetically and chemicall...
Source: Protein Expression and Purification - April 17, 2019 Category: Biochemistry Source Type: research

Expression and purification optimization of an N-terminal Pfs230 transmission-blocking vaccine candidate
Publication date: Available online 10 April 2019Source: Protein Expression and PurificationAuthor(s): Shwu-Maan Lee, Jordan Plieskatt, Seetha Krishnan, Monika Raina, Rakeshkumar Harishchandra, C. Richter KingAbstractIn an effort to control and eventually eliminate malaria, the development of transmission-blocking vaccines has long been sought. However, few antigens have been evaluated in clinical trials, often due to limitations in the expression and purification of the antigen in sufficient yield and quality. Pfs230, a surface antigen of gametocytes, has recently advanced to clinical evaluation as a conjugate vaccine usin...
Source: Protein Expression and Purification - April 11, 2019 Category: Biochemistry Source Type: research

Editorial Board
Publication date: July 2019Source: Protein Expression and Purification, Volume 159Author(s): (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - April 7, 2019 Category: Biochemistry Source Type: research

Strategies for purification of the bacteriophage HK97 small and large terminase subunits that yield pure and homogeneous samples that are functional
Publication date: Available online 4 April 2019Source: Protein Expression and PurificationAuthor(s): Sasha A. Weiditch, Thiago V. Seraphim, Walid A. Houry, Voula KanelisAbstractPackaging the viral genome in the head of double-stranded DNA viruses, such as bacteriophages, requires the activity of a terminase. The bacteriophage terminase consists of a small terminase subunit (TerS), which binds the viral DNA, and a large terminase subunit (TerL) that possesses the ATPase and nuclease activities for packaging the DNA in the phage head. Some phages require additional components for DNA packaging, such as the HNH endonuclease g...
Source: Protein Expression and Purification - April 5, 2019 Category: Biochemistry Source Type: research

CodonWizard – An intuitive software tool for customizable codon optimization in protein expression efforts with graphical user interface
Publication date: Available online 4 April 2019Source: Protein Expression and PurificationAuthor(s): Peter Rehbein, Jannik Berz, Patrick Kreisel, Harald SchwalbeAbstractOptimization of coding sequences to maximize protein expression yield is often outsourced to external service providers during oligonucleotide synthesis and thus remains a black box for many researchers. The presented software tool CodonWizard offers scientists a free, intuitive, easy-to-use software program for customized codon optimization. It provides features for analysis and completely customized modification/optimization of codon usage of any given in...
Source: Protein Expression and Purification - April 5, 2019 Category: Biochemistry Source Type: research

Immobilization of a β-glucosidase and an endoglucanase in ferromagnetic nanoparticles: A study of synergistic effects
Publication date: Available online 31 March 2019Source: Protein Expression and PurificationAuthor(s): Sibeli Carli, Lara Aparecida Buffoni de Campos Carneiro, Richard John Ward, Luana Parras MeleiroAbstractNanoparticles can act as support materials for enzymatic immobilization, introducing a balance of characteristics that modulate the efficiency of biocatalysts, such as specific surface area, resistance to mass transfer and effective enzymatic loading. Magnetic nanoparticles can be easily separated using an external magnetic field, and in this work two recombinant enzymes, the β-glucosidase from Humicola insolens (Bg...
Source: Protein Expression and Purification - April 2, 2019 Category: Biochemistry Source Type: research

The purification of the σFpvI/FpvR20 and σPvdS/FpvR20 protein complexes is facilitated at room temperature
In this study, we describe a purification protocol that resulted in the production of soluble, recombinant His6-σFpvI/FpvR1-67, His6-σFpvI/FpvR1-89, His6-σPvdS/FpvR1-67 and His6-σPvdS/FpvR1-89 protein complexes (where FpvR1-67 and FpvR1-89 are truncated versions of FpvR20) at high purities and concentrations, appropriate for biophysical analyses by circular dichroism spectroscopy and analytical ultracentrifugation. These results showed the proteins to be folded in solution and led to the determination of the affinities of the protein-protein interactions within the His6-σFpvI/FpvR1-67 and His6...
Source: Protein Expression and Purification - March 31, 2019 Category: Biochemistry Source Type: research

Xanthine oxidase of Acinetobacter calcoaceticus RL2-M4: Production, purification and characterization
Publication date: Available online 27 March 2019Source: Protein Expression and PurificationAuthor(s): Monika, Nirmal Kant Sharma, Neerja Thakur Sheetal, Savitri, Tek Chand BhallaAbstractXanthine oxidase (EC 1.17.3.2) is a key enzyme of purine metabolism and has potential applications in food and pharmaceutical industries. In the present study, a new bacterial source of xanthine oxidase i.e. Acinetobacter calcoaceticus RL2-M4 with high oxidase activity was isolated from soil. The culture conditions were optimized with one variable at a time (OVAT) and response surface methodology (RSM) approaches included: a minimal salt me...
Source: Protein Expression and Purification - March 28, 2019 Category: Biochemistry Source Type: research

Molecular cloning and characterization of AlgL17, a new exo-oligoalginate lyase from Microbulbifer sp. ALW1
Publication date: Available online 27 March 2019Source: Protein Expression and PurificationAuthor(s): Zedong Jiang, Yuxi Guo, Xinxia Wang, Hebin Li, Hui Ni, Lijun Li, Anfeng Xiao, Yanbing ZhuAbstractA new alginate lyase gene of algl17 was cloned from an alginate-degrading marine bacterium Microbulbifer sp. ALW1. The gene contained 2220 bp and encoded a 739-amino acid protein classified into the PL-17 family. The recombinant alginate lyase AlgL17 was overexpressed and purified from Escherichia coli BL21 (DE3) with a molecular mass of 84.9 kDa. This enzyme showed activities towards sodium alginate, polyM and alginate oligo...
Source: Protein Expression and Purification - March 28, 2019 Category: Biochemistry Source Type: research

Optimizing high throughput antibody purification by using continuous chromatography media
Publication date: Available online 25 March 2019Source: Protein Expression and PurificationAuthor(s): Rebecca E. Butcher, Genevieve Martin-Roussety, Rebecca A. Bradford, Andrea Tester, Catherine Owczarek, Matthew P. Hardy, Chao-Guang Chen, Georgina Sansome, Louis J. Fabri, Peter M. SchmidtAbstractThe ability to engineer monoclonal antibodies (mAbs) with high specificity made mAbs the fastest growing segment in the drug market. mAbs represent 8 of the top 20 selling drugs with combined sales of more than 57 billion US$ per year. The ability to purify large numbers of mAbs with sufficient yields for initial screening campaig...
Source: Protein Expression and Purification - March 27, 2019 Category: Biochemistry Source Type: research

Efficient expression and secretion of endo-1,4-β-xylanase from Penicillium citrinum in non-conventional yeast Yarrowia lipolytica directed by the native and the preproLIP2 signal peptides
In this study, the xynA gene encoding xylanase A of Penicilium citrinum was successfully synthesized and expressed in Yarrowia lipolytica under the control of the strong constitutive TEF promoter. Native and preproLIP2 secretion signals were used for comparison of the expression and secretion level. The recombinant xylanase was produced as a soluble protein, and the total activity production reached 11 and 52 times higher than the level of activity produced by the fungus P. citrinum native strain, respectively. Maximum activity was observed with the preproLIP2 secretion signal at 180 U/mL. Post translational glycosylation ...
Source: Protein Expression and Purification - March 27, 2019 Category: Biochemistry Source Type: research

Purification of insoluble GST-fused and GST-cleaved Cav1.2 channel fragment by denaturation and renaturation
In this study, we developed a new method to solubilize and purify CT1. GST–CT1 expressed in E. coli was extracted and treated with an inclusion body solubilization and renaturation kit. Then, after adsorption to glutathione Sepharose beads, GST–CT1 was treated with protease to release CT1. However, the cleaved CT1 was insoluble and remained attached to the beads. Therefore, CT1 was treated again with the inclusion body solubilization and renaturation kit. Using this method, GST-CT1 and CT1 were purified with a high yield. GST pull-down experiments showed a dose-dependent interaction between GST–CT1 and ca...
Source: Protein Expression and Purification - March 27, 2019 Category: Biochemistry Source Type: research

Expression and purification of biologically active human granulocyte-macrophage colony stimulating factor (hGM-CSF) using silkworm-baculovirus expression vector system
In this study, we constructed the recombinant baculoviruses for the expression of recombinant hGM-CSF (rhGM-CSF) with two small affinity tags (His-tag and Strep-tag) at the N or C-terminus. Compared to N-tagged rhGM-CSF, C-tagged rhGM-CSF was highly recovered from silkworm hemolymph. The purified rhGM-CSF proteins migrated as a diffuse band and were confirmed to hold N-glycosylations. A comparable activity was achieved when commercial hGM-CSF was tested as a control. Considering the high price of hGM-CSF in the market, our results and strategies using silkworm-baculovirus system can become a great reference for mass produc...
Source: Protein Expression and Purification - March 25, 2019 Category: Biochemistry Source Type: research

Purification of proteins with native terminal sequences using a Ni(II)-cleavable C-terminal hexahistidine affinity tag
Publication date: Available online 22 March 2019Source: Protein Expression and PurificationAuthor(s): Heba A.H. Abd Elhameed, Bálint Hajdu, Ria K. Balogh, Enikő Hermann, Éva Hunyadi-Gulyás, Béla GyurcsikAbstractThe role of the termini of protein sequences is often perturbed by remnant amino acids after the specific protease cleavage of the affinity tags and/or by the amino acids encoded by the plasmid at/around the restriction enzyme sites used to insert the genes. Here we describe a method for affinity purification of a metallonuclease with its precisely determined native termini. First, the g...
Source: Protein Expression and Purification - March 24, 2019 Category: Biochemistry Source Type: research

Expression, purification, and characterization of a metagenomic thioesterase from activated sludge involved in the degradation of acylCoA-derivatives
Publication date: Available online 21 March 2019Source: Protein Expression and PurificationAuthor(s): Jorge Luis Folch-Mallol, Andrés Zárate, Ayixón Sánchez-Reyes, Isabel M. López- LaraAbstractMetagenomic libraries are a novel and powerful approach to seek for pathways involved in xenobiotic degradation, since this technique abolishes the need for cultivating microorganisms that otherwise would be overlooked if they cannot grow on standard laboratory media and conditions. In this paper, we describe the expression, purification and characterization of a novel metagenomic thioesterase which...
Source: Protein Expression and Purification - March 22, 2019 Category: Biochemistry Source Type: research

Expression, purification and characterization of a recombinant antimicrobial peptide Hispidalin in Pichia pastoris
Publication date: Available online 21 March 2019Source: Protein Expression and PurificationAuthor(s): De-Mei Meng, Wen-Juan Li, Lin-Yue Shi, Yu-Jie Lv, Xue-Qing Sun, Jin-Cheng Hu, Zhen-Chuan FanAbstractHispidalin is a novel antimicrobial peptide isolated from the seeds of Benincasa hispida and is reported to have broad antimicrobial activity against various bacterial and fungal pathogens. To produce significant amounts of Hispidalin, a recombinant Hispidalin with an N-terminal 6 × His tag and an enterokinase sequence, for the first time, was successfully expressed in Escherichia coli or Pichia pastoris cell facto...
Source: Protein Expression and Purification - March 22, 2019 Category: Biochemistry Source Type: research

Enhancement of the production of Bacillus naganoensis pullulanase in recombinant Bacillus subtilis by integrative expression
In this study, a universal integrated plasmid was conducted harboring the pul cassette that included the pul gene encoding Bacillus naganoensis pullulanase (PUL), a constitutive promoter, PHpaII, and an extracellular signaling peptide, LipA. This cassette was inserted into the genomes of B. subtilis WB800 and B. subtilis WB600 by double homologous recombination. The pullulanase activity of up to 30.32 U/ml and 18.83 U/ml was achieved for B. subtilis WB800-PHpaII-pul and B. subtilis WB600-PHpaII-pul, respectively, under primary conditions. To further enhance the yield of PUL, the effects of four important factors (inoculum ...
Source: Protein Expression and Purification - March 18, 2019 Category: Biochemistry Source Type: research

Editorial Board
Publication date: June 2019Source: Protein Expression and Purification, Volume 158Author(s): (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - March 16, 2019 Category: Biochemistry Source Type: research

Low binding affinity and reduced complement-dependent cell death efficacy of ofatumumab produced using a plant system (Nicotiana benthamiana L.)
In this study, ofatumumab, a fully human anti-CD20 IgG1κ monoclonal antibody used for its non-cross resistance to rituximab, was expressed in Nicotiana benthamiana, and its affinities and efficacies were compared with those of native ofatumumab produced from CHO cells. Two forms of plant ofatumumab (with or without HDEL-tag) were generated and their production yields were compared. The HDEL-tagged ofatumumab was more expressed in plants than the form without HDEL-tag. The specificity of the target recognition of plant-derived ofatumumab was confirmed by mCherry-CD20-expressing HEK cells via immuno-staining, and the c...
Source: Protein Expression and Purification - March 15, 2019 Category: Biochemistry Source Type: research

The purification of the σFpvI/FpvR and σPvdS/FpvR protein complexes is facilitated at room temperature
In this study, we describe a purification protocol that resulted in the production of soluble, recombinant His6-σFpvI/FpvR1-67, His6-σFpvI/FpvR1-89, His6-σPvdS/FpvR1-67 and His6-σPvdS/FpvR1-89 protein complexes (where FpvR1-67 and FpvR1-89 are truncated versions of FpvR) at high purities and concentrations, appropriate for biophysical analyses by circular dichroism spectroscopy and analytical ultracentrifugation. These results showed the proteins to be folded in solution and led to the determination of the affinities of the protein-protein interactions within the His6-σFpvI/FpvR1-67 and His6-&...
Source: Protein Expression and Purification - March 15, 2019 Category: Biochemistry Source Type: research

Extracellular production of the recombinant bacterial transglutaminase in Pichia pastoris
Publication date: Available online 11 March 2019Source: Protein Expression and PurificationAuthor(s): Aysun Türkanoğlu Özçelik, Fatma Ersöz, Mehmet İnanAbstractMicrobial pro-transglutaminase (pro-MTGase) from Streptomyces mobaraensis was expressed in Pichia pastoris (Komagataella phaffii) under the control of constitutive GAP promoter. The single copy plasmid containing clone was grown in shake flasks to determine the optimum conditions for the production of recombinant pro-MTGase. Three temperature (20 °C, 25 °C, 28 °C) and four pH (5, 6, 7, 7.5) values were evaluated at the shake...
Source: Protein Expression and Purification - March 12, 2019 Category: Biochemistry Source Type: research