Schistosoma mansoni cathepsin D1: Biochemical and biophysical characterization of the recombinant enzyme expressed in HEK293T cells
Publication date: Available online 8 November 2019Source: Protein Expression and PurificationAuthor(s): B.O. Araujo-Montoya, M.R. Senger, B.F. Gomes, G. Harris, R.J. Owens, F.P. Silva-JrAbstractSchistosomes express a variety of aspartyl proteases (APs) with distinct roles in the helminth pathophysiology, among which degradation of host haemoglobin is key, since it is the main amino acid source for these parasites. A cathepsin D-like AP from Schistosoma mansoni (SmCD1) has been used as a model enzyme for vaccine and drug development studies in schistosomes and yet a reliable expression system for readily producing the recom...
Source: Protein Expression and Purification - November 10, 2019 Category: Biochemistry Source Type: research

Removal of half antibody, hole-hole homodimer and aggregates during bispecific antibody purification using MMC ImpRes mixed-mode chromatography
Publication date: Available online 4 November 2019Source: Protein Expression and PurificationAuthor(s): Jiaqin Tang, Xudong Zhang, Tao Chen, Ying Wang, Yifeng LiAbstractDuring recombinant production of asymmetric IgG-like bispecific antibodies (bsAbs), various by-products are often observed due to unbalanced chain expression and incorrect chain pairing. Among them, half antibody and homodimer are found with high frequency. In this work, with a case study we demonstrated that Capto MMC ImpRes mixed-mode chromatography can effectively remove these two by-products as well as antibody aggregates under optimized conditions. Thi...
Source: Protein Expression and Purification - November 6, 2019 Category: Biochemistry Source Type: research

Bacterial overexpression and purification of soluble recombinant human serum albumin using maltose-binding protein and protein disulphide isomerase
Publication date: Available online 4 November 2019Source: Protein Expression and PurificationAuthor(s): Minh Tan Nguyen, Yunseok Heo, Bich Hang Do, Sangki Baek, Chong Jai Kim, Yeon Jin Jang, Weontae Lee, Han ChoeAbstractHuman serum albumin (HSA), the most abundant serum protein in healthy humans, plays important roles in many physiological processes and has wide clinical and research applications. Despite several efforts to obtain recombinant HSA (rHSA) from bacterial and eukaryotic expression systems, a low-cost and high-yield method for rHSA production is not available. The large molecular weight and high disulphide cont...
Source: Protein Expression and Purification - November 6, 2019 Category: Biochemistry Source Type: research

A high yielding IFNAR1 ECD mammalian expression process for use in autoimmune disease drug development
Publication date: Available online 2 November 2019Source: Protein Expression and PurificationAuthor(s): Caroline Kittinger, Arnita Barnes, Alan Hunter, LeeAnn Machiesky, Sandrina Phipps, Anthony Shannon, Robert Stadelman, Susan Wilson, Ellen O'ConnorAbstractInterferon-alpha receptor 1 (IFNAR1) is a target of interest for recombinant biotherapeutics that block the JAK/STAT pathway. This pathway is believed to play a role in many diseases including Hepatitis B and C, Herpes Simplex, Multiple Sclerosis, and other autoimmune disorders. By using IFNAR1 as a target to block Type I IFN from binding to the JAK/STAT pathway and pre...
Source: Protein Expression and Purification - November 3, 2019 Category: Biochemistry Source Type: research

Surface display of classical swine fever virus E2 glycoprotein on gram-positive enhancer matrix (GEM) particles via the SpyTag/SpyCatcher system
In this study, the artificially designed E2-Spy was expressed and glycosylated in Pichia pastoris, and subsequently conjugated with SpyCatcher-PA which was expressed in Escherichia coli. The conjugated E2-Spy-PA was displayed on the surface of GEM particles, generating the E2-Spy-PA-GEM complex. Blocking ELISA analysis and neutralization assays showed that both E2-Spy and E2-Spy-PA-GEM complexes induced high levels of anti-CSFV antibodies in mice. Furthermore, statistical analyses indicated that the E2-Spy-PA-GEM complex exhibited enhanced immunogenicity compared with E2-Spy alone. (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - November 3, 2019 Category: Biochemistry Source Type: research

Optimized expression of classical swine fever virus E2 protein via combined strategy in Pichia pastoris
In this study, two Spy-tagged E2 genes were synthesized in vitro and subcloned into pMCO-AOX vector for intracellular expression in Pichia pastoris after methanol induction. Western blot analysis and semi-quantitative analysis showed that the yield of recombinant E2 protein was improved 17.87 folds by using co-translocational signal peptide cSIG. After the construction of the tandem multiple copy expression vectors, further increase of E2 production was observed by repetitive transforming expression vectors into P. pastoris genome. Finally, the yeast transformants harboring 8 or 16 copies of cSIG-E2-Spy increased the E2 ex...
Source: Protein Expression and Purification - November 3, 2019 Category: Biochemistry Source Type: research

Expression, purification, and structural analysis of the full-length human integral membrane protein γ-sarcoglycan
Publication date: Available online 1 November 2019Source: Protein Expression and PurificationAuthor(s): Michael Jamaladdine, Michael S. Harris, Leshani Liyanage, Gabriel A. CookAbstractMutation of the gene encoding γ-sarcoglycan (SGCG), an integral membrane protein responsible for maintaining the integrity of the muscle cell sarcolemma, results in Limb-Girdle Muscular Dystrophy (LGMD), a congenital disease with no current treatment options. This member of the sarcoglycan glycoprotein family is a vital component of the Dystrophin Complex, which together facilitate normal muscle function. However, very little is known ...
Source: Protein Expression and Purification - November 3, 2019 Category: Biochemistry Source Type: research

Expression and purification of recombinant G protein-coupled receptors: A review
Publication date: Available online 31 October 2019Source: Protein Expression and PurificationAuthor(s): Daniel N. Wiseman, Abigail Otchere, Jaimin H. Patel, Romez Uddin, Naomi L. Pollock, Sarah J. Routledge, Alice J. Rothnie, Cathy Slack, David R. Poyner, Roslyn M. Bill, Alan D. GoddardAbstractGiven their extensive role in cell signalling, GPCRs are significant drug targets; despite this, many of these receptors have limited or no available prophylaxis. Novel drug design and discovery significantly rely on structure determination, of which GPCRs are typically elusive. Progress has been made thus far to produce sufficient q...
Source: Protein Expression and Purification - November 2, 2019 Category: Biochemistry Source Type: research

Molecular characterization of a highly efficient and thermostable phosphoribosyl anthranilate isomerase from Geobacillus thermopakistaniensis
Publication date: Available online 29 October 2019Source: Protein Expression and PurificationAuthor(s): Muhammad Arif, Qamar Bashir, Masood Ahmad Siddiqui, Naeem RashidAbstractPhosphoribosyl anthranilate isomerase is involved in the isomerization of phosphoribosyl anthranilate to 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate. In the present study, trpFGt, a gene encoding phosphoribosyl anthranilate isomerase from Geobacillus thermopakistaniensis, was cloned and expressed in Escherichia coli. The gene product, TrpFGt, was produced in E. coli in soluble and active form. Molecular characterization revealed that recombi...
Source: Protein Expression and Purification - October 31, 2019 Category: Biochemistry Source Type: research

Cloning, characterization and expression analysis of glutathione S-transferase from the Antarctic yeast Rhodotorula mucilaginosa AN5
Publication date: Available online 25 October 2019Source: Protein Expression and PurificationAuthor(s): Cuijuan Shi, Xiaofei Wang, Zijie Xiao, Ruiqi Wang, Yongping Qiao, Guangfeng KanAbstractThe gene for glutathione S-transferase (GST) in Antarctic sea-ice yeast Rhodotorula mucilaginosa AN5 was cloned and expressed in Escherichia coli and named RmGST. Sequence analysis showed that the RmGST gene contained a 843 bp open reading frame, which encoded 280 amino acid residues with a calculated molecular mass of 30.4 kDa and isoelectric point of 5.40. RmGST has the typical C- and N-terminal double domains of glutathione S-tran...
Source: Protein Expression and Purification - October 26, 2019 Category: Biochemistry Source Type: research

Strategies for successful isolation of a eukaryotic transporter
Publication date: Available online 23 October 2019Source: Protein Expression and PurificationAuthor(s): Savvas Saouros, Cristina Cecchetti, Alex Jones, Alexander D. Cameron, Bernadette ByrneAbstractThe isolation of integral membrane proteins for structural analysis remains challenging and this is particularly the case for eukaryotic membrane proteins. Here we describe our efforts to isolate OsBOR3, a boron transporter from Oryza sativa. OsBOR3 was expressed as both full length and a C-terminally truncated form lacking residues 643–672 (OsBOR3Δ1-642). While both express well as C-terminal GFP fusion proteins in ...
Source: Protein Expression and Purification - October 25, 2019 Category: Biochemistry Source Type: research

Recent developments of methyl-labeling strategies in Pichia pastoris for NMR spectroscopy
Publication date: Available online 22 October 2019Source: Protein Expression and PurificationAuthor(s): Meng ZhangAbstractNuclear magnetic resonance (NMR) spectroscopy is a primary structural biology method to characterize protein dynamics in solution. For large macromolecular systems, methyl-labeling in a perdeuterated background significantly improves the relaxation properties, while providing sensitive probes for structure and dynamics analysis. However, how to prepare methyl-labeled proteins, especially for functional eukaryotic proteins, remains to be a major bottleneck in this field. Due to its advantages in eukaryot...
Source: Protein Expression and Purification - October 23, 2019 Category: Biochemistry Source Type: research

Screening and production of an affibody inhibiting the interaction of the PD-1/PD-L1 immune checkpoint
Publication date: Available online 20 October 2019Source: Protein Expression and PurificationAuthor(s): Lei Jing, Juanjuan Liu, Dongxu Cui, Yuyin Li, Zhenxing Liu, Li Tao, Qing Zhao, Aipo DiaoAbstractAn affibody is a 58 amino acids peptide derived from the Z domain of staphylococcal protein A and generally applied in areas such as imaging diagnosis, clinical therapeutics and biotechnology research. To screen for an affibody targeting the immune checkpoint PD-L1, a combinatorial affibody library was generated in yeast using degenerate overlap PCR primers and In-fusion technology. Z-j1 and Z-j2 affibodies targeting the Ig-li...
Source: Protein Expression and Purification - October 22, 2019 Category: Biochemistry Source Type: research

Expression of a Beauveria bassiana chitosanase (BbCSN-1) in Pichia pastoris and enzymatic analysis of the recombinant protein
In this study, a chitosanase gene (BbCSN-1) from Beauveria bassiana, an insect fungal pathogen, was cloned and expressed in Pichia pastoris. The amount of BbCSN-1 in the fermentation broth of P. pastoris gradually increased after induction with methanol from one to 6 d, reaching 398 μg/ml on the 6th day. The molecular characteristics of BbCSN-1 were measured with colloidal chitosan as a substrate. The purified BbCSN-1 exhibited optimum activity at pH 5 and 30 °C and was stable at pH 2–8 and below 40 °C. The Km value of BbCSN-1 was approximately 0.8 mg/ml at 30 °C (pH 6.0). The activity of B...
Source: Protein Expression and Purification - October 20, 2019 Category: Biochemistry Source Type: research

Design, production and purification of a novel recombinant gonadotropin-releasing hormone associated peptide as a spawning inducing agent for fish
Publication date: Available online 16 October 2019Source: Protein Expression and PurificationAuthor(s): Sedigheh Mohammadzadeh, Fatemeh Moradian, Sakineh Yeganeh, Bahram Falahatkar, Sylvain MillaAbstractGnRH is a neuropeptide known to regulate reproduction in vertebrates. The purpose of this study was to design and produce recombinant gonadotropin-releasing hormone associated peptide (rGnRH/GAP) as an alternative of the previous GnRHs and native extracted hormone from tissue, to induce final maturation in fish. Decapeptide as well as GAP area sequences were compared between GnRH1, GnRH2, and mGnRH from Acipenser sp and Hus...
Source: Protein Expression and Purification - October 19, 2019 Category: Biochemistry Source Type: research

Cloning, expression, purification and biochemical characterization of recombinant metallothionein from the white shrimp Litopenaeus vannamei
Publication date: Available online 15 October 2019Source: Protein Expression and PurificationAuthor(s): Jorge Duarte-Gutiérrez, Lilia Leyva-Carrillo, Miguel A. Martínez-Téllez, Rosa O. Méndez-Estrada, Monserrath Felix-Portillo, Gloria Yepiz-PlascenciaAbstractMetallothioneins (MT) are cysteine rich proteins with antioxidant capacity that participate in the homeostasis and detoxification of metals and other cellular processes, and help to counteract the oxidative stress produced by Reactive Oxygen Species (ROS). The production of ROS increases during several stress conditions, including metal into...
Source: Protein Expression and Purification - October 16, 2019 Category: Biochemistry Source Type: research

Editorial Board
Publication date: January 2020Source: Protein Expression and Purification, Volume 165Author(s): (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - October 16, 2019 Category: Biochemistry Source Type: research

Production of potent long-lasting consensus interferon using albumin fusion technology in Pichia pastoris expression system
This study executed the human albumin-fusion technology, a simple and flexible approach to extend the serum circulating half-life of cIFN, because human serum albumin (HSA) has long circulating half-life (19 days) and very minute immunological activities. We integrated the codon-optimized HSA-cIFN fusion gene into Pichia pastoris genome by homologous recombination. The selection of hyper-resistant P. pastoris clone against Zeocin™ achieved a high-level secretory expression (250 mg/L) of fusion protein. HSA-cIFN fusion protein was purified using one-step purification by affinity chromatography with 34% recovery. The...
Source: Protein Expression and Purification - October 10, 2019 Category: Biochemistry Source Type: research

Identification and characterisation of a fluorinase from Actinopolyspora mzabensis
This study describes the identification of a fluorinase from Actinopolyspora mzabensis. Overexpression of the Am-fluorinase in E. coli BL21 (DE3) resulted in the formation of inclusion bodies (IBs). The enzyme was recovered from IBs, solubilised in 8 M urea, and successfully refolded into a biologically active form. Following hydrophobic interaction chromatography,>80 mg of the active fluorinase was obtained at a purity suitable for biocatalytic applications. An additional gel filtration step gave ≥95% pure Am-fluorinase. Using LC-MS/MS, the optimal pH for activity was found at 7.2 while the optimal temperature w...
Source: Protein Expression and Purification - October 7, 2019 Category: Biochemistry Source Type: research

A novel approach for production of an active N-terminally truncated Ulp1 (SUMO protease 1) catalytic domain from Escherichia coli inclusion bodies
Publication date: Available online 4 October 2019Source: Protein Expression and PurificationAuthor(s): Marina Y. Linova, Michael W. Risør, Sanne E. Jørgensen, Zohra Mansour, Jacob Kaya, Jens J. Sigurdarson, Jan J. Enghild, Henrik KarringAbstractThe SUMO fusion system is widely used to facilitate recombinant expression and production of difficult-to-express proteins. After purification of the recombinant fusion protein, removal of the SUMO-tag is accomplished by the yeast cysteine protease, SUMO protease 1 (Ulp1), which specifically recognizes the tertiary fold of the SUMO domain. At present, the expression of...
Source: Protein Expression and Purification - October 5, 2019 Category: Biochemistry Source Type: research

Comparative analysis of fusion tags used to functionalize recombinant antibodies
Publication date: Available online 26 September 2019Source: Protein Expression and PurificationAuthor(s): Gianluca Veggiani, Barbara Giabbai, Marta S. Semrau, Barbara Medagli, Vincenzo Riccio, Gregor Bajc, Paola Storici, Ario de MarcoAbstractRecombinant antibodies can be expressed as fusion constructs in combination with tags which simplify their engineering into reliable and homogeneous immunoreagents by allowing site-specific, 1:1 functionalization. Several tags and corresponding reagents for recombinant protein derivatization have been proposed but benchmarking surveys for the evaluation of their effect on the character...
Source: Protein Expression and Purification - September 27, 2019 Category: Biochemistry Source Type: research

High yield recombinant expression and purification of oncogenic NSD1, NSD2, and NSD3 with human influenza hemagglutinin tag
Publication date: Available online 26 September 2019Source: Protein Expression and PurificationAuthor(s): Yunpeng Shen, Masayo Morishita, Eric di LuccioAbstractThe nuclear receptor-binding SET Domain (NSD) family consists of NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1 histone methyltransferases that are crucial for chromatin remodeling. NSDs are implicated in developmental disorders such as Wolf-Hirschhorn and Sotos syndromes as well as various cancers, including t(4; 14)(p16; q32) myeloma, an incurable cancer in plasma cells. NSDs have been the target of intensive study to understand their biological functions more fully and...
Source: Protein Expression and Purification - September 27, 2019 Category: Biochemistry Source Type: research

pH conditioning is a crucial step in primary recovery - A case study for a recombinant Fab from E. coli
In this study, we investigated pH conditioning during primary recovery for a subsequent cation exchange chromatography (CEX)-based capture of a recombinant Fab produced in E. coli. We showed that pH ≤ 5.0, which is necessary for CEX, led to high product loss due to protein precipitation during cell disruption and pH conditioning. Thus, we developed a procedure that resulted in a 25% increased Fab recovery prior capture based on simple re-arrangement of process steps and the use of a low-cost stabilizing agent. Summarizing, we show the huge potential for simple and cheap improvement of overall downstream process reco...
Source: Protein Expression and Purification - September 26, 2019 Category: Biochemistry Source Type: research

The effect of N-glycosylation on the expression of the tetanus toxin fragment C in Pichia pastoris
Publication date: Available online 21 September 2019Source: Protein Expression and PurificationAuthor(s): Nan Wang, Kevin Yueju Wang, Fangfang Xu, GangQiang Li, DeHu LiuAbstractThe N-glycosylation process that occurs in the Pichia pastoris protein expression system can have a significant effect on the yield of heterologous glycoproteins secreted from the yeast. The basis of the effect of N-glycosylation on yield, however, has not been elucidated. In order to investigate the effect of N-glycosylation on heterologous protein production, site-directed mutation was performed on five potential N-glycosylation sites of the tetan...
Source: Protein Expression and Purification - September 22, 2019 Category: Biochemistry Source Type: research

A bacterial endo-β-1,4-glucuronan lyase, CUL-I from Brevundimonas sp. SH203, belonging to a novel polysaccharide lyase family
In this study, the gene encoding CUL-I was cloned, and the recombinant enzyme was heterologously expressed in Escherichia coli. The predicted CUL-I protein is composed of 426 amino acid residues and includes a putative 21-amino acid signal peptide. The recombinant CUL-I specifically depolymerized β-1,4-glycoside linkages of cellouronate, and its mode of action was endo-type, like the native CUL-I. Sequence analysis showed CUL-I has no similarity to previously known polysaccharide lyases (PLs), indicating that CUL-I should be classified into a novel PL family. (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - September 21, 2019 Category: Biochemistry Source Type: research

Bax expression is optimal at low oxygen tension and constant agitation
Publication date: Available online 19 September 2019Source: Protein Expression and PurificationAuthor(s): Yi He, Yong Chen, Daniel L. Morris, Duck-Yeon Lee, Nico TjandraAbstractBax is a pro-apoptosis protein that translocates from the cytosol to the mitochondrial membrane upon initiation of programed cell death. Bax subsequently disrupts the mitochondria membrane, resulting in the release of cytochrome C which activates the downstream caspases. The structure of inactive Bax has been solved, but despite intensive investigation, the mechanism by which it regulates apoptosis is not established. The low yield of Bax expression...
Source: Protein Expression and Purification - September 21, 2019 Category: Biochemistry Source Type: research

A short peptide fragment of the vascular endothelial growth factor as a novel ligand for bevacizumab purification
Publication date: Available online 19 September 2019Source: Protein Expression and PurificationAuthor(s): Gabriela R. Barredo, Silvana L. Giudicessi, María C. Martínez Ceron, Soledad L. Saavedra, Santiago Rodriguez, Lucas Filgueira Risso, Rosa Erra-Balsells, Gustavo Mahler, Fernando Albericio, Osvaldo Cascone, Silvia A. CamperiAbstractBevacizumab is a vascular endothelial growth factor (VEGF)-directed monoclonal antibody (mAb) used for the treatment of several human cancers. Given that bevacizumab is administered intravenously, it must have extremely high purity, which is achieved by purification with protein...
Source: Protein Expression and Purification - September 21, 2019 Category: Biochemistry Source Type: research

Recombinant expression and purification of AF1q and its interaction with T-cell Factor 7
Publication date: Available online 18 September 2019Source: Protein Expression and PurificationAuthor(s): Nazimuddin Khan, Jino Park, William L. Dean, Robert D. Gray, William Tse, Donghan Lee, T. Michael SaboAbstractThe protein ALL1 fused from chromosome 1q (AF1q) is overexpressed in a variety of cancers and acts to activate several signaling pathways that lead to oncogenesis. For example, AF1q has been shown to interact with T-cell Factor 7 (TCF7; also known as TCF1) from the Wnt/β-catenin pathway resulting in the transcriptional activation of the CD44 and the enhancement of breast cancer metastasis. Despite the impo...
Source: Protein Expression and Purification - September 19, 2019 Category: Biochemistry Source Type: research

Efficient production of wild-type lipase B from Candida antarctica in the cytoplasm of Escherichia coli
Publication date: Available online 12 September 2019Source: Protein Expression and PurificationAuthor(s): Lisette Van Tassel, Antti Moilanen, Lloyd W. RuddockAbstractCandida antarctica lipase B (CalB) is a very efficient catalyst and is used in a wide range of industries from food flavour to pharmaceutical, and biodiesel manufacturing. It has a high degree of enantioselective and regioselective substrate specificity and is stable over a wide range of biophysical conditions including pH, temperature and solvent conditions. High-level expression of biologically active wild-type CalB has been problematic, partly due to foldin...
Source: Protein Expression and Purification - September 13, 2019 Category: Biochemistry Source Type: research

Corrigendum to ‘Cloning and purification of an anti-thrombotic, chimeric Staphylokinase in Pichia pastoris’ [Protein Expr. Purif. 162 (2019) 1–8]
Publication date: Available online 11 September 2019Source: Protein Expression and PurificationAuthor(s): Vandana, Satish Kantipudi, Neeraj Maheshwari, Sheetal Sharma, Girish Sahni (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - September 13, 2019 Category: Biochemistry Source Type: research

High-volume shake flask cultures as an alternative to cellbag technology for recombinant protein production in the baculovirus expression vector system (BEVS)
Publication date: Available online 10 September 2019Source: Protein Expression and PurificationAuthor(s): Ciarán N. Cronin (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - September 11, 2019 Category: Biochemistry Source Type: research

Expression, purification and crystallization of an SLC16 monocarboxylate transporter family homologue specific for l-lactate
Publication date: Available online 9 September 2019Source: Protein Expression and PurificationAuthor(s): Sara Bonetti, Stephan Hirschi, Patrick D. BosshartAbstractl-lactate plays an important role as metabolite and signaling molecule in eukaryotes and bacteria. Monocarboxylate transporters (MCTs) of the SLC16 solute carrier family are responsible for the transport of l-lactate across eukaryotic and bacterial cell membranes. Here we report an efficient protocol for the expression and purification of an SLC16 family homologue in milligram amounts. The purified protein is stable and can thus be used for biochemical and struct...
Source: Protein Expression and Purification - September 10, 2019 Category: Biochemistry Source Type: research

Development of BioRad NGC and GE ÄKTA pure systems for highly automated three column protein purification employing tandem affinity, buffer exchange and size exclusion chromatography
Publication date: Available online 7 September 2019Source: Protein Expression and PurificationAuthor(s): Dwight Winters, Mai Tran, Daniel Yoo, Kenneth WalkerAbstractAffinity purification, such as Protein A (Pro A) followed by size exclusion chromatography (SEC) remains a popular method to obtain research scale proteins. With the need for higher throughput protein production increasing for discovery research, there is substantial interest in the automation of complex protein purification processes, which often start with a Pro A step followed by SEC. However, the harsh elution conditions from Pro A based chromatography can ...
Source: Protein Expression and Purification - September 8, 2019 Category: Biochemistry Source Type: research

Editorial Board
Publication date: December 2019Source: Protein Expression and Purification, Volume 164Author(s): (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - September 5, 2019 Category: Biochemistry Source Type: research

Optimization of recombinant maize CDKA;1 and CycD6;1 production in Escherichia coli by response surface methodology
Publication date: Available online 31 August 2019Source: Protein Expression and PurificationAuthor(s): Andrea A.E. Méndez, Liliana B. Pena, Lucrecia M. Curto, Marcelo D. Sciorra, Rita M. Ulloa, Sara M. Garza Aguilar, Jorge M. Vázquez Ramos, Susana M. GallegoAbstractThe complex formed by the cyclin-dependent kinase A (CDKA) and cyclin D is responsible for the G1-S transition in the plant cell cycle. Maize (Zea mays L) CDKA; 1 and CycD6; 1 were cloned and expressed in E. coli. The present study describes the optimization of both proteins production using a statistical approach known as response surface methodol...
Source: Protein Expression and Purification - September 1, 2019 Category: Biochemistry Source Type: research

Enzymatic properties of a multi-specific β-(1,3)-glucanase from Corallococcus sp. EGB and its potential antifungal applications
Publication date: Available online 27 August 2019Source: Protein Expression and PurificationAuthor(s): Jie Zhou, Jianhao Chen, Zhoukun Li, Xianfeng Ye, Weiliang Dong, Min Jiang, Yan Huang, Zhongli CuiAbstractThe lamC gene encoding a novel β-(1,3)-glucanase was cloned from Corallococcus sp. EGB and successfully expressed in the industrial yeast Pichia pastoris. The mature protein without the initial 26 residues of signal peptide, designated LamC27, was found to be composed of fascin-like module and laminarinase-like catalytic module. The purified recombinant enzyme (rLamC27) with a calculated molecular mass of 45.3 k...
Source: Protein Expression and Purification - August 29, 2019 Category: Biochemistry Source Type: research

Improving expression of thermostable trehalase from Myceliophthora sepedonium in Aspergillus niger mediated by the CRISPR/Cas9 tool and its purification, characterization
Publication date: Available online 27 August 2019Source: Protein Expression and PurificationAuthor(s): Liangbo Dong, Dou Yu, Xiaotong Lin, Bin Wang, Li PanAbstractTrehalase catalyzes the conversion of one molecule of trehalose into two glucose molecules. The trehalase TreM from thermophilic fungus Myceliophthora sepedonium was expressed in Aspergillus niger via traditional homologous recombination with trehalase activity of 406.44 U/mL. The multi-copy knock-in expression strategy mediated by the CRISPR/Cas9 tool was used to improve the production of the TreM trehalase in Aspergillus niger, which was up to 1943.06 U/mL with...
Source: Protein Expression and Purification - August 29, 2019 Category: Biochemistry Source Type: research

Expression, purification and stabilization of human serotonin transporter from E. coli
Publication date: Available online 20 August 2019Source: Protein Expression and PurificationAuthor(s): Daniel Worms, Barbara Maertens, Jan Kubicek, Udaya Kumar Tiruttani Subhramanyam, Jörg LabahnAbstractThe serotonin transporter belongs to the family of sodium-chloride coupled neurotransmitter transporter and is related to depression in humans. It is therefore an important drug target to support treatment of depression. Recently, structures of human serotonin transporter in complex with inhibitor molecules have been published. However, the production of large protein amounts for crystallization experiments remains a b...
Source: Protein Expression and Purification - August 22, 2019 Category: Biochemistry Source Type: research

Preparation and characterization of a highly soluble Aβ1-42 peptide variant
Publication date: Available online 16 August 2019Source: Protein Expression and PurificationAuthor(s): Marcia A. LeVatte, Matthias Lipfert, Carol Ladner-Keay, David S. WishartAbstractAlzheimer's disease (AD) is a progressive neurological disease marked by the accumulation and deposition of misfolded amyloid beta or Abeta (Aβ) peptide. Two species of Aβ peptides are found in amyloid plaques, Aβ1-40 and Aβ1-42, with the latter being the more amyloidogenic of the two. Understanding how and why Aβ peptides misfold, oligomerize and form amyloid plaques requires a detailed understanding of their structur...
Source: Protein Expression and Purification - August 17, 2019 Category: Biochemistry Source Type: research

Expression and characterization of an extremely thermophilic 1,4-α-glucan branching enzyme from Rhodothermus obamensis STB05
Publication date: Available online 14 August 2019Source: Protein Expression and PurificationAuthor(s): Zhe Wang, Chenhao Xin, Caiming Li, Zhengbiao Gu, Li Cheng, Yan Hong, Xiaofeng Ban, Zhaofeng LiAbstractA gene encoding 1,4-α-glucan branching enzyme (GBE, EC 2.4.1.18) from the extremely thermophilic bacterium Rhodothermus obamensis STB05 was successfully cloned and expressed in Escherichia coli. Extracellular expression of the recombinant enzyme (R.o-GBE) was achieved with a yield of 1080 mg/L. Then it was purified and further characterized biochemically. R.o-GBE was optimally active at pH 7.0 and 65 °C. It ...
Source: Protein Expression and Purification - August 16, 2019 Category: Biochemistry Source Type: research

A new metal affinity NCTR25 tag as a better alternative to the His-tag for the expression of recombinant fused proteins
Publication date: Available online 13 August 2019Source: Protein Expression and PurificationAuthor(s): Weitong Pan, Yan Wang, Nan WangAbstractHis-tagging is commonly used in fusion protein production, but the His-tag is usually prohibited in medicinal proteins and must be removed. A fragment (NCTR25-tag) truncated from the N-terminus of human copper transporter 1 was tested for feasibility as a replacement for the His-tag in fusion proteins. The NCTR25-tag and His-tag were separately fused to the transthyretin (TTR) protein, and the expression, affinity purification, refolding and stability of the two kinds of fusions were...
Source: Protein Expression and Purification - August 14, 2019 Category: Biochemistry Source Type: research

High-level heterologous expression of the human transmembrane sterol Δ8,Δ7-isomerase in Pichia pastoris
Publication date: December 2019Source: Protein Expression and Purification, Volume 164Author(s): Hongmin Cai, Hebang Yao, Tingting Li, Yannan Tang, Dianfan LiAbstractRecombinant expression of human membrane proteins in large quantities remains a major challenge. Expression host is an important variable to screen for high-level production of membrane proteins. Using the green fluorescent protein (GFP) as a reporter, we screened the expression of a human multi-pass membrane protein called sterol Δ8-Δ7 isomerase in three different hosts: Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. The expressi...
Source: Protein Expression and Purification - August 10, 2019 Category: Biochemistry Source Type: research

Editorial Board
Publication date: November 2019Source: Protein Expression and Purification, Volume 163Author(s): (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - August 7, 2019 Category: Biochemistry Source Type: research

High-level heterologous expression of the transmembrane human sterol Δ8,Δ7-isomerase in Pichia pastoris
Publication date: Available online 2 August 2019Source: Protein Expression and PurificationAuthor(s): Hongmin Cai, Hebang Yao, Tingting Li, Yannan Tang, Dianfan LiAbstractRecombinant expression of human membrane proteins in large quantities remains a major challenge. Expression host is an important variable to screen for high-level production of membrane proteins. Using the green fluorescent protein (GFP) as a reporter, we screened the expression of a human multi-pass membrane protein called sterol Δ8-Δ7 isomerase in three different hosts: Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. The exp...
Source: Protein Expression and Purification - August 3, 2019 Category: Biochemistry Source Type: research

Expression, purification and characterisation of Trypanosoma congolense metacaspase 5 (TcoMCA5) - a potential drug target for animal African trypanosomiasis
Publication date: Available online 1 August 2019Source: Protein Expression and PurificationAuthor(s): Lauren E-A. Eyssen, Theresa H.T. CoetzerAbstractThe metacaspases (MCAs) are attractive drug targets for the treatment of African trypanosomiasis as they are not found in the metazoan kingdom and their action has been implicated in cell cycle and cell death pathways in kinetoplastid parasites. Here we report the biochemical characterisation of MCA5 from T. congolense. Upon recombinant expression in E. coli, autoprocessing is evident, and MCA5 further autoprocesses when purified using nickel affinity chromatography, which we...
Source: Protein Expression and Purification - August 3, 2019 Category: Biochemistry Source Type: research

Heterologous expression, purification and biochemical characterization of a new endo-1,4-β-xylanase from Rhodothermaceae bacterium RA
In this study, a xylanase gene (1140 bp) that encoded 379 amino acids from the bacterium was cloned and expressed in Escherichia coli BL21(DE3). Based on InterProScan, this enzyme XynRA1 contained a GH10 domain and a signal peptide sequence. XynRA1 shared low similarity with the currently known xylanases (the closest is 57.2–65.4% to Gemmatimonadetes spp.). The purified XynRA1 achieved maximum activity at pH 8 and 60 °C. The protein molecular weight was 43.1 kDa XynRA1 exhibited an activity half-life (t1/2) of 1 h at 60 °C and remained stable at 50 °C throughout the experiment. However, it was N...
Source: Protein Expression and Purification - August 3, 2019 Category: Biochemistry Source Type: research

Expression, purification, and in vitro characterization of kinase domain of NtGCN2 from tobacco
In this study, the kinase domain of Nicotiana tabacum GCN2 (NtGCN2) was inserted into the pET15b vector for prokaryotic expressionin Escherichia coli BL21-CodonPlus-(DE3)-RIPL after induction by 0.5 mmol L−1 IPTG for 13 h at 16 °C. The soluble protein was collected and purified by Ni2+-NTA agarose column, anion exchange, and molecular sieve, and the purified proteinwas used for kinase assays and the preparation of a polyclonal antibody. Enzyme-linked immunosorbent assay results showed that the titer of the antiserum was 1:520K. Western blot analysis showed that the prepared antibody reacted with GCN2 in...
Source: Protein Expression and Purification - August 1, 2019 Category: Biochemistry Source Type: research

Characterization and expression in Pichia pastoris of a raw starch degrading glucoamylase (GA2) derived from Aspergillus flavus NSH9
Publication date: Available online 26 July 2019Source: Protein Expression and PurificationAuthor(s): Kazi Muhammad Rezaul Karim, Ahmad Husaini, Ngieng Ngui Sing, Tasmia Tasnim, Fazia Mohd Sinang, Hasnain Hussain, Md Anowar Hossain, Hairul RoslanAbstractA pH and thermostable glucoamylase encoding gene having starch-binding domain (SBD) from Aspergillus flavus NSH9 was successfully identified, isolated, and expressed in Pichia pastoris GS115 to produce recombinant glucoamylase (rGA2). The full-length glucoamylase gene (2039 bp) and cDNA (1839 bp) isolated was found to encode for 612 amino acid residues in the open reading fr...
Source: Protein Expression and Purification - July 27, 2019 Category: Biochemistry Source Type: research

Purification and partial biochemical characterization of recombinant lactate dehydrogenase 1 (LDH-1) of the white shrimp Litopenaeus vannamei
We report the production of soluble LDH-1 by testing different pHs in the buffers used to lyse the bacterial cells before the purification step and the characterization of the purified protein to show that the cDNA indeed codes for a functional and active protein. The recombinant native protein is a homotetramer of approximately 140 kDa composed by 36 kDa subunits and has higher affinity for pyruvate than for lactate. LDH-1 has an optimum pH of 7.5 and is stable between pH 8.0 and 9.0; pH data analysis showed two pKa values of 6.1 ± 0.15 and 8.8 ± 0.15 suggesting a histidine and asparagine, respec...
Source: Protein Expression and Purification - July 27, 2019 Category: Biochemistry Source Type: research

Sodium caprylate induced precipitation post protein a chromatography as an effective means for host cell protein clearance
In this study, we replaced CA with its sodium salt – sodium caprylate (SC). For the five mAbs studied, SC has been shown to be equally effective as CA at precipitating HCPs. As the salt form has a higher solubility, SC stock solution with relatively high concentration can be easily prepared, which facilitates its adding to the Protein A elution pool. Thus, this study not only confirms the effectiveness of CA/SC-induced HCP precipitation but also provides a more convenient way to integrate this method into the downstream process. (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - July 27, 2019 Category: Biochemistry Source Type: research