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Protein Expression and Purification

Søren Heissel, Jakob Bunkenborg, Max Per Kristiansen, Anne Fich Holmbjerg, Marie Grimstrup, Ejvind Mørtz, Thomas Kofoed, Peter Højrup
Recombinantly expressed biopharmaceutical proteins often undergo a series of purification steps with the aim of removing contaminating material. Depending on the application of the protein, there are various requirements for the degree of purity, but host cell proteins (HCPs) will in general remain in small amounts. LC-MS has emerged as an orthogonal technique, capable of providing detailed information regarding the individual proteins. The aim of this case study was to characterize the HCPs associated with a biopharmaceutical protein, provided by Statens Serum Institut (DK), which is used in the field of tuberculosis and has not previously been studied by LC-MS...
March 8, 2018: Protein Expression and Purification
De-Mei Meng, Yu-Jie Lv, Jing-Fang Zhao, Qing-Yan Liu, Lin-Yue Shi, Jun-Ping Wang, Yong-Hai Yang, Zhen-Chuan Fan
VpDef is a novel defensin isolated from the clam Venerupis philippinarum. Previously it was expressed in Escherichia coli; however, the E. coli-derived recombinant VpDef did not show effective antimicrobial activity against Staphyloccocus aureus or the Gram-negative bacteria tested. As such, the goal of this study was to design, express, and purify a recombinant VpDef (rVpDef) in Pichia pastoris and to determine its antibacterial potency and stability. A 6.9 KDa rVpDef was successfully expressed as a secreted peptide in P...
March 6, 2018: Protein Expression and Purification
Wen Zhu, Guihua Gong, Jie Pan, Shu Han, Wei Zhang, Youjia Hu, Liping Xie
Human serum albumin (HSA) has been extensively used in a series of clinical care settings for nearly seven decades. However, the broad application of this protein is seriously limited by its short supply. In this work, the codon sequence of HSA was cloned under the control of the alcohol oxidase 1 promoter (AOX1) and expressed as a secretory protein in Pichia pastoris. A recombinant strain displaying the highest HSA yield was selected by screening for resistance to the highest concentration of antibiotic G418...
March 5, 2018: Protein Expression and Purification
Mengmeng Zhao, Ping Li, Yi Xie, Xiang Liu, Lin Cheng, Tingyan Liu, Lijun Kong, Oumei Wang, Fengchan Han
The erl mouse is a mouse model of nonsyndromic autosomal recessive deafness (DFNB12) on the C57BL/6J background. This project was carried out to express the first two ectodomains of cadherin 23 (CDH23 EC1+2) of erl mice in Escherichia coli and identify the Ca2+ -binding ability of the recombinant protein. DNA sequences of CDH23 EC1+2 from wild type and erl mice were synthesized and cloned into pBV220 plasmids. Recombinant plasmids were transformed into Escherichia coli and expression of CDH23 EC1+2 was induced by increasing the temperature from 30 °C to 42 °C...
February 24, 2018: Protein Expression and Purification
Ario de Marco
The possibility of successfully applying nanomaterials such as biosensors or nanoparticles in diagnostics and therapy is critically dependent on the capacity to optimize their target recognition selectivity and their ability to be delivered minimizing off-side accumulation. Biological macromolecules possess the necessary specificity and for this reason have been often coupled to nanomaterials. However, such process is not straightforward because it often induces structural alterations of the involved macromolecules, in most of the cases proteins or antibodies the functions of which can be hampered when single amino acids are modified...
February 24, 2018: Protein Expression and Purification
Parvaneh Katoli, Adarsh Godbole, Michael J Romanowski, Kirk Clark, Erik Meredith, Veronica Saenz-Vash, Y Karen Wang, Nancy Lewicki, Andrew A Nguyen, Jeffrey M Lynch
Myocilin (MYOC) is a secreted protein found in human aqueous humor (AH) and mutations in the MYOC gene are the most common mutation observed in glaucoma patients. Human AH analyzed under non-reducing conditions suggests that MYOC is not normally found in a monomeric form, but rather is predominantly dimeric. Although MYOC was first reported almost 20 years ago, a technical challenge still faced by researchers is an inability to isolate full-length MYOC protein for experimental purposes. Herein we describe two methods by which to isolate sufficient quantities of human full-length MYOC protein from mammalian cells...
February 20, 2018: Protein Expression and Purification
Mariana Chesini, Evelyn Wagner, Diego J Baruque, Carolina E Vita, Sebastián F Cavalitto, Pablo D Ghiringhelli, Natalia L Rojas
Exoinulinases-enzymes extensively studied in recent decades because of their industrial applications-need to be produced in suitable quantities in order to meet production demands. We describe here the production of an acid-stable recombinant inulinase from Aspergillus kawachii in the Pichia pastoris system and the recombinant enzyme's biochemical characteristics and potential application to industrial processes. After an appropriate cloning strategy, this genetically engineered inulinase was successfully overproduced in fed-batch fermentations, reaching up to 840 U/ml after a 72-h cultivation...
February 15, 2018: Protein Expression and Purification
Sehyeon Ji, Hyosuk Yun, Gwansik Park, Hye Jung Min, Chul Won Lee
Rattusin is an α-defensin-related peptide isolated from the small intestine of rats. The primary sequence of linear rattusin is composed of 31 amino acids containing five cysteines with a unique spacing pattern. It forms a homodimeric scaffold in which the primary structure occurs in an antiparallel fashion formed by five intermolecular disulfide (SS) bonds. Rattusin is a highly potent antibiotic, which not only exhibits broad-spectrum antimicrobial activity, but also maintains its antimicrobial activity at physiological salt concentrations...
February 14, 2018: Protein Expression and Purification
Liangcheng Jiao, Qinghua Zhou, Zhixin Xu, Li Xu, Yunjun Yan
Rhizopus oryzae lipase (ROL) is an important industrial enzyme limited in application due to its low production in native strains. Here, we used a new combined strategy to overexpress ROL in Pichia pastoris. An efficient method based on bio-brick was developed to construct a series of vectors harboring different copy numbers of ROL gene cassettes, which were then transformed into P. pastoris GS115 to generate a strain with specific copy numbers of ROL. An optimized gene-dosage recombinant strain of GS115/pAOα-5ROL 11# harboring five copies of ROL was screened, revealing production of the highest activity (2700 U/mL), which was 8-fold higher than that of the strain harboring one copy...
February 13, 2018: Protein Expression and Purification
José Manoel Wanderley Duarte Neto, Maria Carolina de Albuquerque Wanderley, Carolina de Albuquerque Lima, Ana Lúcia Figueiredo Porto
A new set of applications can be achieved when using high stability proteases. Industrially, high costs can be related to production medium and purification process. Magnetic nanoparticles have been successfully used for rapid and scalable purification. In this work, azocasein were immobilized on magnetite nanoparticles and applied in a single step purification of protease produced by Penicillium aurantiogriseum using soybean flour medium, and the new purified enzyme was characterized. Glutaraldehyde activated nanoparticles were used in azocasein immobilization and then incubated with dialyzed 60-80% saline precipitation fraction of crude extract for purification...
February 12, 2018: Protein Expression and Purification
Yunsoo Kim, Nils Benning, Kasey Pham, Anna Baghdadi, Gino Caruso, Megan Colligan, Allyssa Grayson, Alexis Hurley, Nick Ignatoski, Sandra Mcclure, Kelsey Mckaig, Emily Neag, Cole Showers, Alexis Tangalos, Jessica Vanells, Kaillathe Padmanabhan, Zachary F Burton
Homology threading is a powerful technology for generating structural models based on homologous structures. Here we use threading to generate four complex RNA polymerase models. The models appear to be as useful as x-ray crystal structures or cryo-electron microscopy structures to support research projects.
February 11, 2018: Protein Expression and Purification
Takahiro Nonaka, Noriko Tsurui, Teruhisa Mannen, Yoshimi Kikuchi, Kentaro Shiraki
This paper describes a new pH-responsive peptide tag that adds a protein reversible precipitation and redissolution character. This peptide tag is a part of a cell surface protein B (CspB) derived from Corynebacterium glutamicum. Proinsulin that genetically fused with a peptide of N-terminal 6, 17, 50, or 250 amino acid residues of CspB showed that the reversible precipitation and redissolution depended on the pH. The transition occurred within a physiological and narrow pH range. A CspB50 tag comprising 50 amino acid residues of N-terminal CspB was further evaluated as a representative using other pharmaceutical proteins...
February 9, 2018: Protein Expression and Purification
Shogo Oki, Takahiro Nonaka, Kentaro Shiraki
Protein purification using non-chromatographic methods is a simple technique that avoids costly resin. Recently, a cell surface protein B (CspB) tag has been developed for a pH-responsive tag for protein purification by solid-liquid separation. Proteins fused with the CspB tag show reversible insolubilization at acidic pH that can be used in solid-liquid separation for protein purification. However, brown-color impurities from co-precipitation hamper further analysis of the target proteins. In this study, we investigated the effect of additives on the co-precipitation of CspB-tagged Teriparatide (CspB50TEV-Teriparatide) expressed in Corynebacterium glutamicum and associated impurities...
February 6, 2018: Protein Expression and Purification
Anvarsadat Kianmehr, Morteza Oladnabi, Abdolkarim Mahrooz, Javad Ansari, Rahman Mahdizadeh
Diaphorases are flavin-containing enzymes with potential applications in biotransfomation reactions, biosensor design and in vitro diagnostic tests. In this paper, we present recombinant expression, characterization and medium optimization of a lipoamide dehydrogenase (DLD) with NADH-dependent diaphorase activity from a Lysinibacillus sp. strain. DLD encoding sequence showed an open reading frame of 1413-bp encoding a 470 amino acid chain. Lysinibacillus sp. DLD catalyzed the NADH-dependent reduction of electron acceptors and exhibited diaphorase activity...
February 2, 2018: Protein Expression and Purification
Kuo-Ming Yu, Johnson Yiu-Nam Lau, Manson Fok, Yuk-Keung Yeung, Siu-Ping Fok, Felix Shek, Wing-Tak Wong, Qui-Lim Choo
Current source of recombinant human interleukin-11 (rhIL-11) is isolated from a fusion protein expressed by E. coli that requires additional enterokinase to remove linked protein, resulting in product heterogeneity of N-terminal sequence. Due to lack of glycosylation, rhIL-11 is suitable to be expressed by yeast cells. However, the only available yeast-derived rhIL-11 presents an obstacle in low production yield, as well as an unamiable process, such as the use of reverse-phase chromatography employing plenty of toxic organic solvents...
February 2, 2018: Protein Expression and Purification
Petr Rathner, Michael Stadlbauer, Christoph Romanin, Marc Fahrner, Isabella Derler, Norbert Müller
We report a new NMR-scale purification procedure for two recombinant wild type fragments of the stromal interaction molecule 1 (STIM1). This protein acts as a calcium sensor in the endoplasmic reticulum (ER) and extends into the cytosol accumulating at ER - plasma membrane (PM) junctions upon calcium store depletion ultimately leading to activation of the Orai/CRAC channel. The functionally relevant cytosolic part of STIM1 consists of three coiled coil domains, which are mainly involved in intra- and inter-molecular homomeric interactions as well as coupling to and gating of CRAC channels...
February 1, 2018: Protein Expression and Purification
Mark T Agasid, Xuemin Wang, Yiding Huang, Colleen M Janczak, Robert Branström, S Scott Saavedra, Craig A Aspinwall
The inwardly rectifying K+ (Kir) channel, Kir6.2, plays critical roles in physiological processes in the brain, heart, and pancreas. Although Kir6.2 has been extensively studied in numerous expression systems, a comprehensive description of an expression and purification protocol has not been reported. We expressed and characterized a recombinant Kir6.2, with an N-terminal decahistidine tag, enhanced green fluorescent protein (eGFP) and deletion of C-terminal 26 amino acids, in succession, denoted eGFP-Kir6...
January 30, 2018: Protein Expression and Purification
Ritika Chauhan, Vinita Chauhan, Mula Kameshwar Rao, Dilip Chaudhary, Sameer Bhagyawant, Ram Kumar Dhaked
Botulinum neurotoxins (BoNTs) are the most toxic biological substances known. Their potential use as biological warfare agent results in their classification as category A biowarfare agent by Centers for Disease Control and Prevention (CDC), USA. Presently, there are no approved detection system and pharmacological treatments for BoNT intoxication. Although a toxoid vaccine is available for immuno-prophylaxis, vaccines cannot reverse the effect of pre-translocated toxin. Direct handling of the live BoNTs for developing detection and therapeutics may pose fatal danger...
January 30, 2018: Protein Expression and Purification
Othman Rechiche, Jeffrey E Plowman, Duane P Harland, T Verne Lee, J Shaun Lott
Keratin-associated proteins (KAPs) were identified 70 years ago in wool follicles. KAPs are encoded by several multi-gene families and are classified into three different groups: ultra-high sulfur (UHS), high sulfur (HS) and high glycine-tyrosine (HGT). KAPs are the major constituent of the matrix between the hair keratin intermediate filaments (IFs), and stabilise hair structure by extensive disulfide bonding. However, detailed molecular structural information is lacking for KAPs and for KAP interactions with IFs...
January 29, 2018: Protein Expression and Purification
Hisayo Shimizu, Masataka Nakagawa, Nemuri Todaka, Keitaro Imaizumi, Yasunori Kurosawa, Toshiaki Maruyama, C J Okumura, Takashi Shibata, Yosuke Tanaka, Yoshinori Sato, Yasuo Ono, Teruo Akuta
Rabbit monoclonal antibodies (mAbs) have many advantages over mouse antibodies in biological research and diagnostics applications because they exhibit high affinity and specificity. However, the methods of recombinant rabbit mAb production have not been optimized to the same extent as techniques used to produce mouse and human mAbs. In this study, we sought to optimize the production of a recombinant rabbit mAb against human plexin domain containing protein 2 (PLXDC2), a known cell surface antigen, by culturing HEK293-6E cells transfected with antibody-encoding genes at two different temperatures and by purifying the end-product by three different chromatography methods...
January 26, 2018: Protein Expression and Purification
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