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Protein Expression and Purification

Keshav V, Achilonu I, Dirr Hw, Kondiah K
PbrD is a lead (II) binding protein encoded by the pbr lead resistance operon found exclusively in Cupriavidus metallidurans CH34. Its ability to sequester Pb(II) shows potential for it to be developed as a biosorbent for Pb in the bioremediation of contaminated wastewaters. In this study the pbrD gene from C. metallidurans CH34 was transformed and overexpressed in Escherichia coli BL21 (DE3) using the pET32 Xa/Lic vector. Optimal expression of recombinant (r)PbrD (∼50 kDa) was achieved post-induction with IPTG within inclusion bodies (IBs)...
February 15, 2019: Protein Expression and Purification
G J Mc Callum, M B Arregui, I Smith, L F Bracco, F Wolman, O Cascone, A M Targovnik, M V Miranda
Expression of recombinant proteins with baculovirus-infected insect larvae is a scarcely investigated alternative in comparison to that in insect cell lines, a system with growing popularity in the field of biotechnology. The aim of this study was to investigate the chromatographic behavior and physicochemical properties of the proteome of Rachiplusia nu larvae infected with recombinant Autographa californica multiple nucleopolyhedrosis virus (AcMNPV), in order to design rational purification strategies for the expression of heterologous proteins in this very complex and little-known system, based on the differential absorption between target recombinant proteins and the system's contaminating ones...
February 14, 2019: Protein Expression and Purification
Qiusheng Zhang, Binglian Xu, Jiajia Pan, Danyang Liu, Ruoxian Lv, Dongchun Yan
Grass carp reovirus (GCRV) is one of the most serious pathogens threatening grass carp (Ctenopharyngodon idellus) production and results in high mortality in China. VP7 from GCRV is involved in viral infection and could be suitable for developing vaccines for the control of GCRV infection. To obtain a genetically engineered vaccine and a plant-based oral vaccine and to evaluate their immune efficacy as an oral vaccine against GCRV, cholera toxin B subunit (CTB) of Vibrio cholerae fused to VP7 (CTB-VP7) was transformed into BL21(DE3) for expression...
February 9, 2019: Protein Expression and Purification
Rasmus Kock Flygaard, Beatrice Malacrida, Patrick Kiely, Lasse Bohl Jenner
Human elongation factor 2 is the translocase that is responsible for the movement of tRNA from the A-to P and P- to E-site on the ribosome during the elongation phase of translation. Being a vital factor of protein biosynthesis, its function is highly controlled and regulated. It has been implicated in numerous diseases and pathologies, and as such it is important to have a source for isolated pure and active protein for biomedical and biochemical studies. Here we report development of a purification protocol for native human elongation factor 2 from HEK-293S cells...
February 8, 2019: Protein Expression and Purification
Rachel Orlomoski, Aaron Bogle, Jeanmarie Loss, Rylee Simons, Jacqueline M Dresch, Robert A Drewell, Donald E Spratt
Homeodomain transcription factors (HD TFs) are a large class of evolutionarily conserved DNA binding proteins that contain a basic 60-amino acid region required for binding to specific DNA sites. In Drosophila melanogaster, many of these HD TFs are expressed in the early embryo and control transcription of target genes in development through interaction with cis-regulatory modules. Previous studies where some of the Drosophila HD TFs were purified required the use of strong denaturants (i.e. 6M urea) and multiple chromatography columns, making the downstream biochemical examination of the isolated protein difficult...
February 7, 2019: Protein Expression and Purification
Artur P G Dingeldein, Mikael J Lindberg, Jörgen Åden, Xueyin Zhong, Raphael Stoll, Gerhard Gröbner
Mitochondria-mediated apoptosis (programmed cell death) involves a sophisticated signaling and regulatory network that is regulated by the Bcl-2 protein family. Members of this family have either pro- or anti-apoptotic functions. An important pro-apoptotic member of this family is the cytosolic Bax. This protein is crucial for the onset of apoptosis by perforating the mitochondrial outer membrane (MOM). This process can be seen as point of no return, since disintegration of the MOM leads to the release of apotogenic factors such as cytochrome c into the cytosol triggering the activation of caspases and subsequent apoptotic steps...
February 6, 2019: Protein Expression and Purification
Hannah Welte, Michael Kovermann
High resolution NMR spectroscopy is a seminal method in modern structural biology to obtain insights into proteins' structure, dynamics and function at dilute condition as well as in a cell-like environment or even intracellularly. Usually, 1 H, 15 N or 13 C nuclei are predominantly used for the characterization of the protein of interest. These measurements are limited due to the wealth of chemical shifts and background signals arising from all molecules present in the NMR test tube. On top of that, the protein under study has to be isotopically enriched in nitrogen and/or carbon nuclei enabling to overcome the inherently low natural abundance of 13 C and 15 N NMR active isotopes...
February 6, 2019: Protein Expression and Purification
Marcin Zieliński, Agnieszka Romanik-Chruścielewska, Diana Mikiewicz, Natalia Łukasiewicz, Iwona Sokołowska, Jarosław Antosik, Agnieszka Sobolewska-Ruta, Anna Bierczyńska-Krzysik, Piotr Zaleski, Andrzej Płucienniczak
The number of people with diabetes is estimated to be over 370 million, in 2030 it will increase to 552 million. In Poland, the number of people with diabetes is estimated to be 3.5 million (9.1%). According to the estimates of the International Diabetes Federation, the percentage of patients in the adult Polish population will increase to around 11% over the next 20 years. Despite the appearance of insulin analogues on the pharmaceutical market, insulin delivery is still the most effective method of pharmacotherapy in cases of extremely high hyperglycemia...
February 5, 2019: Protein Expression and Purification
He Zhu, Lin Zhao, Zemin Li, Biyan Wen, Chuangnan Qiu, Mengmiao Liu, Zhimin Xu, Shuzhuang Hu, Huangjin Li
Epidermal growth factor receptor (EGFR) is an effective target for the treatment of many epithelial cancers. However, EGFR inhibitors have low clinical response rates and are prone to drug resistance arising from mutations and heterodimerization of EGFR. Therefore, targeting the highly conserved dimer interface of EGFR may be an effective strategy for improving the clinical response of anti-EGFR therapies. Nanobodies have significant advantages over conventional antibodies in terms of size, solubility, stability and cost-effectiveness...
February 5, 2019: Protein Expression and Purification
Hanmei Li, Zeeshan Ali, Xiaolong Liu, Jiang Li, Yongjun Tang, Jianguo Dai
The development of antibiotic-resistant bacteria has become a major public health problem, prompting the search for alternative solutions. Tachyplesin I (TP-I) is an antimicrobial peptide, which exhibits potent and broad-spectrum activities against bacteria, fungi, viruses, and tumor cells. However, limited amounts of TP-I produced in horseshoe crab restrict its large-scale use. In order to solve this problem, a eukaryotic expression system of Pichia pastoris with high TP-I expression was constructed by gene engineering...
January 31, 2019: Protein Expression and Purification
E V Schmalhausen, M S Shumkov, V I Muronetz, V K Švedas
The goal of the present work was to produce glyceraldehyde-3-phospate dehydrogenase from M. tuberculosis in E. coli cells in soluble and catalytically active form and to elaborate a method for the purification of the recombinant enzyme. The His-tagged recombinant enzyme (Mtb-GAPDH_His) was shown to be inactive and insoluble. The untagged enzyme (Mtb-GAPDH) was catalytically active and exhibited higher solubility. Mtb-GAPDH was purified from the cell extract using ammonium sulfate fractionation and ion-exchange chromatography...
January 30, 2019: Protein Expression and Purification
Nafiseh Alsadat Seyed Hosseini Fin, Mohammad Barshan-Tashnizi, Seyed Mehdi Sajjadi, Saemeh Asgary, Nazanin Mohajerani, Hasan Mirzahoseini
The secretory production of heterologous proteins in E. coli has revolutionized biotechnology. Efficient periplasmic production of foreign proteins in E. coli often requires a signal peptide to direct proteins to the periplasm. However, the presence of attached signal peptide does not guarantee periplasmic expression of target proteins. Overproduction of auxiliary proteins, such as chaperones can be a useful approach to enhance protein export. In the current study, three chaperone plasmid sets, including GroEL-GroES (GroELS), Dnak-Dnaj-GrpE (DnaKJE), and trigger factor (TF), were coexpressed in E...
January 29, 2019: Protein Expression and Purification
Erika L Crowley, Steven P Rafferty
NMR is an important method in the structural and functional characterization of proteins, but such experiments typically require isotopic labelling because of the low natural abundance of the nuclei of interest. Isotope-labelled protein for NMR experiments is typically obtained from IPTG-inducible bacterial expression systems in a minimal media that contains labelled carbon or nitrogen sources. Optimization of expression conditions is crucial yet challenging; large amounts of labelled protein are desired, yet protein yields are lower in minimal media, while the labelled precursors are expensive...
January 29, 2019: Protein Expression and Purification
Mateja Rebernik, Brigita Lenarčič, Marko Novinec
Cathepsin C is a tetrameric lysosomal protease that acts as a dipeptidyl-peptidase due to the presence of the exclusion domain that is unique among papain-like cysteine proteases. Here we describe a recombinant form of cathepsin C lacking its exclusion domain (CatCΔEx) produced in bacterial expression system (E. coli). CatCΔEx is a monomer with endoprotease activity and affinity for hydrophobic residues such as Phe, Leu or Pro, but not Val, in the P2 position. As opposed to cathepsin C, it does not require chloride ions for its activity...
January 28, 2019: Protein Expression and Purification
Jie Qiao, Ce Dong, Xinping Wang, Yi Liu, Lixin Ma
Human lipopolysaccharide (LPS) binding protein (LBP) is a ∼60 kDa glycosylated protein that mediates potent innate immune against invading Gram-negative bacteria by recognition of LPS in their outer membranes. To date, there is no method for efficient production of bioactive LPS-free LBP at sufficient amounts through prokaryotic expression system. Here we present a simple approach for rapid preparation of human LBP from a LPS-eliminated E. coli strain named ClearColi BL21 (DE)3. Combined with the usage of an ultra-high-affinity CL7/Im7 purification system, we achieved one-step purification of recombinant human LBP with over 90% purity at a yield of ∼4 mg/L when using LB culture medium...
January 25, 2019: Protein Expression and Purification
Narsing Rao Saroja, Anil H Shyam Mohan, D Srividya, K Supreetha
A putrescine monooxygenase from Shewanella putrefaciens 95 (SpPMO) is the initial enzyme catalyzing the hydroxylation of putrescine to N-hydroxyl putrescine, the precursor for the synthesis of a siderophore putrebactin was identified. This PMO clustered together with known characterized NMOs from Shewanella baltica, Bordetella pertussis, Erwinia amylovora, Streptomyces sp. Gordonia rubripertincta, Pseudomonas aeruginosa and outgrouped from Escherichia coli, Nocardia farcinica, and Rhodococcus erythropolis. The deduced SpPMO protein showed 53% and 36% sequence identity with other characterized bacterial NMOs from Erwinia amylovora and Gordonia rubripertincta respectively...
January 14, 2019: Protein Expression and Purification
Apinun Kanpiengjai, Kridsada Unban, Thu-Ha Nguyen, Dietmar Haltrich, Chartchai Khanongnuch
Lactobacillus pentosus BA-7 and L. pentosus QA1-5 are tannin-tolerant lactic acid bacteria that were isolated from Miang, a traditional fermented tea-leaf found in northern Thailand and a tannin-rich substrate. Tannase encoding genes were isolated, cloned and overexpressed in Escherichia coli BL21(DE3). The recombinant tannase was produced with production yields of 40 and 39 KU/L for LpTanBA-7 and LpTanQA1-5, respectively. Both revealed the same molecular weight of 50 kDa as estimated by SDS-PAGE and were optimally active under alkaline pH conditions that are distinct from the reported bacterial tannase...
January 9, 2019: Protein Expression and Purification
Xiao-Dan Li, Li-Juan Zhou, Cheng Zhao, Lu Lu, Nan-Nan Niu, Jia-Xin Han, Kai-Hong Zhao
Naturally-occurring orange carotenoid protein (OCP) is synthesized in cyanobacteria and red algae for photoprotection. Holo-OCP can be produced with three plasmids in E. coli, which needs two inducers (arabinose and isopropyl β-D-thiogalactoside) to initiate two processes: one for generation of carotenoid and the other for generation of apo-OCP, so takes about two days. Afterwards, a two-plasmid method using two plasmids in E. coli is established, in which E. coli cells are induced only by isopropyl β-D-thiogalactoside, so can yield different holo-OCPs from several cyanobacteria within three days...
January 7, 2019: Protein Expression and Purification
Yotsombat Akkharapimon, Hasegawa Tae, Mino Kohei, Takata Goro
The isolated Rhizobium sp. S10 was identified as D-glucoside 3-dehydrogenase (G3DH) producing microbes. Therefore, the gene encoding for G3DH from Rhizobium sp. S10 was cloned and overexpressed in Escherichia coli strain JM109 as a soluble enzyme. The yield flavoenzyme contains 1686 bp of a complete open reading frame and encodes for 561 amino acid residues. The flavin adenine dinucleotide binding region locates in the N-terminal region of G3DH. The recombinant G3DH (rG3DH) was purified with specific activity of 38...
January 7, 2019: Protein Expression and Purification
Masao Tokunaga, Tsutomu Arakawa, Yuhei Tokunaga, Yasushi Sugimoto, Matsujiro Ishibashi
Insoluble expression of intrinsically soluble proteins with native activity is potentially a promising alternative to soluble expression of folded protein or insoluble expression of unfolded protein requiring refolding. Here, we attempted to express highly soluble halophilic His-rich metal binding protein (HP) as insoluble inclusion bodies with native metal-binding activity using insolubilizing nona-peptide (Ins), GILQINSRW, derived from hen egg white lysozyme (His-InsHP). About 80% of expressed His-InsHP was localized in inclusion bodies in Na-phosphate/NaCl buffer, pH 7...
January 4, 2019: Protein Expression and Purification
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