Enhancing the yield of human erythropoietin in Aspergillus niger by introns and CRISPR-Cas9
Publication date: Available online 15 January 2020Source: Protein Expression and PurificationAuthor(s): Uriel Rojas-Sánchez, Alberto Cristian López-Calleja, Blanca E. Millán-Chiu, Francisco Fernández, Achim M. Loske, Miguel A. Gómez-LimAbstractAspergillus niger has been employed to produce heterologous proteins due to its high capacity for expression and secretion; nevertheless, expression levels of human proteins have been modest. We were interested in investigating whether A. niger can express and secret human erythropoietin (HuEPO) at high yields. Our strategy was to combine the presence of introns with CRISPR-Cas9...
Source: Protein Expression and Purification - January 17, 2020 Category: Biochemistry Source Type: research

Production of membrane proteins in industry: The example of GPCRs
Publication date: Available online 14 January 2020Source: Protein Expression and PurificationAuthor(s): James C. Errey, Cédric Fiez-VandalAbstractWhereas membrane proteins make up ∼23% of the human proteome, it is estimated that membrane proteins constitute more than 60% of current drug targets. With membrane proteins forming such a high percentage of drug targets relative to their abundance within the proteome, it is little wonder that drug companies need to rapidly access high quality membrane proteins for their drug discovery process. Newly devised technologies, such as rapid low-cost gene synthesis, novel detergents...
Source: Protein Expression and Purification - January 15, 2020 Category: Biochemistry Source Type: research

Heterologous production of porcine antimicrobial peptide PR-39 in Escherichia coli using SUMO and intein fusion systems
In this study, we presented two different expression systems for the production of PR-39 in E. coli; one in fusion with Intein-Chitin binding domain (CBD) and another in fusion with SUMO accompanied by polyhistidine affinity tag. Both were cloned in the NdeI-XhoI sites of pET-17b and transformed to E. coli BL21 (DE3) pLysS. Recombinant bacteria were cultured and induced with 0.4 mM IPTG at 30 °C. Expression and purification of target proteins were confirmed by Tricine- SDS-PAGE and dot blot analysis. Recovery of 250 μg PR-39/L from SUMO fusion system and 280 μg PR-39/L from the intein fusion system was achieved....
Source: Protein Expression and Purification - January 14, 2020 Category: Biochemistry Source Type: research

Novel strategy for expression and characterization of rabies virus glycoprotein
In this study, we solved this problem by replacing the original signal peptide of rabies virus G protein with the one from the heavy chain of human IgG. The expression levels of recombinant G protein dramatically increased from a few μg/L to 50 mg/L in the culture supernatants. The identity of the recombinant G protein was confirmed by western blotting using both 6XHis mAb 6E2 and rabies G protein mAb 7G3. The correct conformation of the recombinant G protein was shown by using rabies virus neutralizing antibodies. In addition, the recombinant G protein had immune-reactivities with mice sera raised against rabies vaccin...
Source: Protein Expression and Purification - January 5, 2020 Category: Biochemistry Source Type: research

Expression, purification and characterization of 5′-nucleotidase from caterpillar fungus by efficient genome-mining
In this study, a 5′-NT gene was identified and mined from genomic DNA of caterpillar fungus, which was 1968 bp in length and encoded 656 amino acid residues. The recombinant 5′-NT was first time heterologously expressed in Pichia pastoris GS115, subsequently purified and functionally characterized. The optimal reaction temperature for 5′-NT was 35 °C, and it retained 52.8% of its residual activity after incubation at 50 °C for 1 h. The optimal reaction pH was 6.0 and it exhibited high activity over a neutral pH range. Furthermore, 5′-NT exhibited excellent Km (1.107 mM), Vmax (0.113 μmol/mg·min) and k...
Source: Protein Expression and Purification - January 2, 2020 Category: Biochemistry Source Type: research

Heteroexpression and biochemical characterization of thermostable citrate synthase from the cyanobacteria Anabaena sp. PCC7120
Publication date: Available online 27 December 2019Source: Protein Expression and PurificationAuthor(s): Ya-Dong Ge, Lu-Lu Jiang, Shao-Lin Hou, Feng-Zhi Su, Peng Wang, Gen ZhangAbstractThe present study recombinantly expressed a citrate synthase from cyanobacteria Anabaena sp. PCC7120 (AnCS) in Escherichia coli and characterized its enzymatic activity. The molecular mass of native AnCS was 88,533.1 Da containing two 44,162.7 Da subunits. Recombinant AnCS revealed the highest activity at pH 9.0 and 25 °C. AnCS displayed high thermal stability with a half-life time (t1/2) of approximately 6.5 h at 60 °C, which ...
Source: Protein Expression and Purification - December 28, 2019 Category: Biochemistry Source Type: research

Bacterial sugar-binding protein as a one-step affinity purification tag on dextran-containing resins
Publication date: Available online 26 December 2019Source: Protein Expression and PurificationAuthor(s): Brett Kinrade, Peter L. Davies, Tyler D.R. VanceAbstractMarinobacter hydrocarbonoclasticus is an oil-eating bacterium that possesses a large adhesion protein (MhLap) with the potential to bind extracellular ligands. One of these ligand-binding modules is the ∼20-kDa PA14 domain (MhPA14) that has affinity for glucose-based carbohydrates. Previous studies showed this sugar-binding domain is retained on dextran-based size-exclusion resins during chromatography, requiring the introduction of glucose or EDTA to remove the ...
Source: Protein Expression and Purification - December 27, 2019 Category: Biochemistry Source Type: research

Editorial Board
Publication date: March 2020Source: Protein Expression and Purification, Volume 167Author(s): (Source: Protein Expression and Purification)
Source: Protein Expression and Purification - December 24, 2019 Category: Biochemistry Source Type: research

Refolding and purification of cGMP-grade recombinant human neurturin from Escherichia coli inclusion bodies
Publication date: Available online 19 December 2019Source: Protein Expression and PurificationAuthor(s): Guoling Xi, Reza Esfandaria, Chester Bittencourt Sacramento, Hani Jouihan, Arun Sharma, Robert Roth, Thomas LinkeAbstractNeurturin is a potent neurotrophic factor that has been investigated as a potential therapeutic agent for the treatment of neurodegenerative diseases, including Parkinson's disease, and, more recently, for the treatment of type II diabetes. However, purification of neurturin for clinical applications has been hampered by its low solubility in aqueous solutions. Here we describe the development of a sc...
Source: Protein Expression and Purification - December 21, 2019 Category: Biochemistry Source Type: research

Cloning, expression and characterization of a thermo-alkali-stable xylanase from Aspergillus oryzae LC1 in Escherichia coli BL21(DE3)
Publication date: Available online 12 December 2019Source: Protein Expression and PurificationAuthor(s): Nisha Bhardwaj, Vijay Kumar Verma, Venkatesh Chaturvedi, Pradeep VermaAbstractIn the present investigation, cloning and overexpression of xylanase (XynF1), the main xylanase of A. oryzae LC1, was performed in prokaryotic system E. coli BL21(DE3) to produce recombinant xylanase with high titer of specific activity (1037.3 U/mg), which was 9.3-fold higher than the native strain. Further, the recombinant XynF1 of size 37 kDa was purified using Ni2+-NTA resins followed by cation exchange chromatography, which showed an 1....
Source: Protein Expression and Purification - December 13, 2019 Category: Biochemistry Source Type: research

Immunization of rabbits with recombinant Clostridium perfringens alpha toxins CPA-C and CTB-CPA-C in a bicistronic design expression system confers strong protection against challenge
In this study, we used an E. coli system for the efficient production of recombinant proteins and developed a bicistronic design (BCD) expression construct consisting of two copies of the C-terminal (247–370) domain of the alpha toxin (CPA-C) in the first cistron, followed by Cholera Toxin B (CTB) linked with another two copies of CPA-C in the second cistron that is controlled by a single promoter. Rabbits were immunized twice with purified proteins (rCPA-C rCTB-CPA-C) produced in the BCD expression system, with an inactivated recombinant E. coli vaccine (RE), C. perfringens formaldehyde-inactivated alpha toxoid (FA-CPA)...
Source: Protein Expression and Purification - December 5, 2019 Category: Biochemistry Source Type: research

Purification and biochemical characteristics of a novel fructosyltransferase with a high FOS transfructosylation activity from Aspergillus oryzae S719
In this study, the novel fructosyltransferase (FTase) from Aspergillus oryzae strain S719 was successfully purified and characterized. The specific activity of the final purified material was 4200 mg−1 with purification ratio of 66 times and yield of 26%. The molecular weight of FTase of A. oryzae S719 was around 95 kDa by SDS-PAGE, which was identified as a type of FTase by Mass Spectrometry (MS). The purified FTase had optimum temperature and pH of 55 °C and 6.0, respectively. The FTase showed to be stable with more than 80% of its original activity at room temperature after 12 h and maintaining activity above...
Source: Protein Expression and Purification - December 4, 2019 Category: Biochemistry Source Type: research

Non-structural protein 1 from Japanese encephalitis virus expressed in E. coli retains its molecular weight and immunogenicity
Publication date: Available online 29 November 2019Source: Protein Expression and PurificationAuthor(s): Jae-Won Choi, Hyo-Ji Eom, Hak Yong KimAbstractJapanese encephalitis virus (JEV) is a member of the Flavivirus genus and has recently attracted attention as a high-risk pathogen in the Asia-Pacific region, with up to 30% mortality in the afflicted patients. Recent outbreaks of flavivirus-associated infections around the world have put the focus on non-structural protein 1 (NS1) as a candidate for diagnostic and vaccine researches on flaviviruses. Although the JEV NS1 protein has been expressed in eukaryotic cells, attemp...
Source: Protein Expression and Purification - November 29, 2019 Category: Biochemistry Source Type: research

Characterization of a hyperphosphorylated variant of G protein-coupled receptor kinase 5 expressed in E. coli
Publication date: Available online 29 November 2019Source: Protein Expression and PurificationAuthor(s): Tyler S. Beyett, Qiuyan Chen, Emily J. Labudde, Joseph Krampen, Prateek V. Sharma, John J.G. TesmerAbstractG protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors in humans and regulate numerous physiological processes through the activation of heterotrimeric G proteins. GPCR kinases (GRKs) selectively phosphorylate active GPCRs, which promotes arrestin binding, receptor internalization, and initiation of alternative signaling pathways. GRK5 is a representative member of one of three GRK sub...
Source: Protein Expression and Purification - November 29, 2019 Category: Biochemistry Source Type: research

Expression and purification of codon-optimized cre recombinase in E. coli
Publication date: Available online 27 November 2019Source: Protein Expression and PurificationAuthor(s): Srividya D, Anil H. Shyam Mohan, Saroja Narsing RaoAbstractThe presence of antibiotic resistance genes in genetically modified bacteria raises a regulatory concern in the production of therapeutic proteins and additionally reduces the number of plasmids available for propagation in a cell. Cre recombinase from bacteriophage P1, involved in Cre/loxP mechanism is one of the widely used systems for selectable marker gene removal. We have overexpressed codon-optimized cre gene in pColdIV and pET28a(+) vector systems and pur...
Source: Protein Expression and Purification - November 28, 2019 Category: Biochemistry Source Type: research