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Development of a biochemical and biophysical suite for integral membrane protein targets: A review
Publication date: Available online 26 November 2019Source: Protein Expression and PurificationAuthor(s): Matthias Haffke, Myriam Duckely, Christian Bergsdorf, Veli-Pekka Jaakola, Binesh ShresthaAbstractThe generation of integral membrane proteins (IMPs) in heterologous systems and their characterization remains a major challenge in biomedical research. Significant efforts have been invested both in academia and in the pharmaceutical industry to establish technologies for the expression, isolation and characterization of IMPs. Here we summarize some of the key aspects, which are important to support structure-based drug des...
Source: Protein Expression and Purification - November 28, 2019 Category: Biochemistry Source Type: research

High-level expression and characterization of the thermostable leucine aminopeptidase Thelap from the thermophilic fungus Thermomyces lanuginosus in Aspergillus niger and its application in soy protein hydrolysis
In this study, we obtained the highly active and thermostable leucine aminopeptidases screened from the thermophilic fungi Thermomyces lanuginosus, Talaromyces thermophilus, and Malbranchea cinnamomea. The activity of the recombinant leucine aminopeptidase Thelap was significantly increased to 2771.5 U/mL, as mediated by the CRISPR/Cas9 tool. The recombinant Thelap was easily purified from fermentation broth by Ni-affinity chromatography, and the specific activity of the purified Thelap was increased to 7449.6 U/mg. The recombinant Thelap showed optimal activity at pH 8.5 and 75 °C and remained above 70% of the maximum ...
Source: Protein Expression and Purification - November 22, 2019 Category: Biochemistry Source Type: research

Expression and purification of recombinant mouse CRISP4 using a baculovirus system
In this study, we report a three-step purification protocol for expression and purification of mouse CRISP4 protein in High Five™ cells using a baculovirus expression system. Recombinant mouse CRISP4 was recognized by western blotting and structurally characterized using Circular Dichroism (CD). Using the protocol described herein, we generated high yields of soluble and correctly folded recombinant mouse CRISP4. (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - November 22, 2019 Category: Biochemistry Source Type: research

The impact of a His-tag on DNA binding by RNA polymerase alpha-C-terminal domain from Helicobacter pylori
In this study, we demonstrate that a His-tag impacts DNA complex formation by the C-terminal domain of the ??-subunit (??CTD) of Helicobacter pylori RNA polymerase in a metal-dependent fashion. The ??CTD was purified with a cleavable His-tag, and complex formation between αCTD, the nickel-responsive metalloregulator HpNikR, and DNA was investigated using electrophoretic mobility shift assays. An interaction between His-tagged ??CTD (His??CTD) and the HpNikR-DNA complex was observed; however, this interaction was not observed upon removal of the His-tag. Further analysis revealed that complex formation between HisαCTD and...
Source: Protein Expression and Purification - November 20, 2019 Category: Biochemistry Source Type: research

Retinol binding protein IV purified from Escherichia coli using intein-mediated cleavage as a suitable replacement for serum sources
Publication date: Available online 19 November 2019Source: Protein Expression and PurificationAuthor(s): Chandler B. Est, Regina M. MurphyAbstractRetinol binding protein IV (RBP) functions as the principal carrier of retinol (Vitamin A) in the blood, where RBP circulates bound to another serum protein, transthyretin. Isolation of pure RBP from the transthyretin complex in human serum can be difficult, but expression of RBP in recombinant systems can circumvent these purification issues. Human recombinant RBP has previously been successfully expressed and purified from E. coli, but recovery of active protein typically requi...
Source: Protein Expression and Purification - November 20, 2019 Category: Biochemistry Source Type: research

N-terminal fusion of the N-terminal domain of bacterial enzyme I facilitates recombinant expression and purification of the human RNA demethylases FTO and Alkbh5
Publication date: Available online 15 November 2019Source: Protein Expression and PurificationAuthor(s): Balabhadra Khatiwada, Jeffrey A. Purslow, Eric S. Underbakke, Vincenzo VendittiAbstractVarious fusion tags are commonly employed to increase the heterologous expression and solubility of aggregation-prone proteins within Escherichia coli. Herein, we present a protocol for efficient recombinant expression and purification of the human RNA demethylases Alkbh5 and FTO. Our method incorporates a novel fusion tag (the N-terminal domain of bacterial enzyme I, EIN) that dramatically increases the solubility of its fusion partn...
Source: Protein Expression and Purification - November 16, 2019 Category: Biochemistry Source Type: research

New method for immobilising diverse proteins onto cubic micro-protein polyhedrin crystals
Publication date: Available online 14 November 2019Source: Protein Expression and PurificationAuthor(s): Haruna Yuasa, Eiji Kotani, Hajime Mori, Keiko TakakiAbstractCypovirus is an insect virus that is encapsulated in stable cubic protein crystals composed of polyhedrin protein produced in virus-infected cells. Molecular technology developed over the last decade is now able to immobilise proteins of interest on polyhedrin crystals. Modified polyhedrin crystals can be used in cell cultures for implantation in animals and vaccines, among other applications. However, this technique does not work for some proteins. Here, we de...
Source: Protein Expression and Purification - November 15, 2019 Category: Biochemistry Source Type: research

Editorial Board
Publication date: February 2020Source: Protein Expression and Purification, Volume 166Author(s): (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - November 12, 2019 Category: Biochemistry Source Type: research

Improving the heterologous expression of human β-defensin 2 (HBD2) using an experimental design
Publication date: Available online 9 November 2019Source: Protein Expression and PurificationAuthor(s): Ligia Luz Corrales-García, Leobardo Serrano-Carreón, Gerardo CorzoAbstractAt present, expressing antimicrobial peptides in bacterial models is considered a routine lab bench work. However, low expression yields of these types of proteins are usually obtained. In this work, the antimicrobial peptide human β-defensin 2 (HBD2) was obtained in low expression yields in Escherichia coli BL21(DE3). To improve the expression yields of HBD2, some variables such as cell density, temperature, and length of induction, as well as ...
Source: Protein Expression and Purification - November 10, 2019 Category: Biochemistry Source Type: research

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