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Affinity purification recombinant protein

Tomoyuki Hatano, Salvatore Alioto, Emanuele Roscioli, Saravanan Palani, Scott T Clarke, Anton Kamnev, Juan Ramon Hernandez-Fernaud, Lavanya Sivashanmugam, Bernardo Chapa-Y-Lazo, Alexandra M E Jones, Robert C Robinson, Karuna Sampath, Masanori Mishima, Andrew D McAinsh, Bruce L Goode, Mohan K Balasubramanian
Actins are major eukaryotic cytoskeletal proteins, which perform many important cell functions, including cell division, cell polarity, wound healing, and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively presently for biochemical studies of actin cytoskeleton from other organisms / cell types. Here we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Pichia pastoris Actin is expressed as a fusion with the actin-binding protein thymosin β4 and purified using an affinity tag introduced in the fusion...
March 13, 2018: Journal of Cell Science
E Quintero-Troconis, N Buelvas, C Carrasco-López, M R Domingo-Sananes, L González-González, R Ramírez-Molina, L Osorio, A Lobo-Rojas, A J Cáceres, P A Michels, H Acosta, W Quiñones, J L Concepción
Purification of enolase (ENO) from the cytosol of Trypanosoma cruzi indicated that it may interact with at least five other proteins. Two of them were identified as metallocarboxypeptidase-1 (TcMCP-1) and a putative acireductone dioxygenase (ARDp). Subcellular localization studies confirmed the presence of ARDp in the cytosol, as is the case for ENO and TcMCP-1. Analysis of the ARDp sequence showed that this protein has two domains, an N-terminal ARD and a C-terminal TRP14 (thioredoxin-related protein) domain...
March 9, 2018: Biochimica et Biophysica Acta
Alejandro Saettone, Jyoti Garg, Jean-Philippe Lambert, Syed Nabeel-Shah, Marcelo Ponce, Alyson Burtch, Cristina Thuppu Mudalige, Anne-Claude Gingras, Ronald E Pearlman, Jeffrey Fillingham
BACKGROUND: The chromatin remodelers of the SWI/SNF family are critical transcriptional regulators. Recognition of lysine acetylation through a bromodomain (BRD) component is key to SWI/SNF function; in most eukaryotes, this function is attributed to SNF2/Brg1. RESULTS: Using affinity purification coupled to mass spectrometry (AP-MS) we identified members of a SWI/SNF complex (SWI/SNFTt ) in Tetrahymena thermophila. SWI/SNFTt is composed of 11 proteins, Snf5Tt , Swi1Tt , Swi3Tt , Snf12Tt , Brg1Tt , two proteins with potential chromatin-interacting domains and four proteins without orthologs to SWI/SNF proteins in yeast or mammals...
March 9, 2018: Epigenetics & Chromatin
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
Jeffrey A Lawton, Noelle A Prescott, Ping X Lawton
We have developed an integrated, project-oriented curriculum for undergraduate molecular biology and biochemistry laboratory courses spanning two semesters that is organized around the ldhA gene from the yogurt-fermenting bacterium Lactobacillus bulgaricus, which encodes the enzyme d-lactate dehydrogenase. The molecular biology module, which consists of nine experiments carried out over eleven sessions, begins with the isolation of genomic DNA from L. bulgaricus in yogurt and guides students through the process of cloning the ldhA gene into a prokaryotic expression vector, followed by mRNA isolation and characterization of recombinant gene expression levels using RT-PCR...
March 7, 2018: Biochemistry and Molecular Biology Education
Prasannan V Anu, Madathiparambil G Madanan, Ananthakrishnan J Nair, Gangaprasad A Nair, Govinda Pillai M Nair, Perumana R Sudhakaran, Padikara K Satheeshkumar
Oligopeptidases are enzymes involved in the degradation of short peptides (generally less than 30 amino acids in size) which help pathogens evade the host defence mechanisms. Leptospira is a zoonotic pathogen and causes leptospirosis in mammals. Proteome analysis of Leptospira revealed the presence of oligopeptidase A (OpdA) among other membrane proteins. To study the role of oligopeptidase in leptospirosis, the OpdA of L. interrogans was cloned and expressed in Escherichia coli with a histidine tag (His-tag)...
March 3, 2018: Molecular Biotechnology
Rofida M Abd El-Fatah, Noha M Mesbah, Dina M Abo-Elmatty, Khaled A Aly
The human pathogen Staphylococcus aureus encodes the ESAT6-like Secretion System (ESS). The ESS pathway secretes pathogenic substrates such as EsxA, EsxB, EsxC, EsxD and EssD that mediate staphylococcal establishment in persistent abscess lesions. The biochemical behavior of these substrates is not fully understood. EsxC is species-specific lysine-rich homodimer that lacks recognizable topogenic sequence. Studies have shown that EsxC is required for the secretion of other substrates, thereby revealing its biomedical importance...
February 27, 2018: Toxicon: Official Journal of the International Society on Toxinology
Parminder K Ajji, Shailendra P Sonkar, Ken Walder, Munish Puri
Balsamin, a type I ribosome-inactivating protein (RIP), has been shown to inhibit HIV-1 replication at the translation step. Our recent studies have shown that balsamin also possess anti-tumor, antibacterial and DNase-like activity, however, the amount of natural balsamin in Momordica balsamina seeds is limited and preclinical studies require large quantities of pure, bioactive balsamin. Therefore, in this study, we cloned the balsamin gene, expressed it in E. coli BL21(DE3) strain and purified it by nickel affinity chromatography...
February 19, 2018: International Journal of Biological Macromolecules
Diego A Díaz-Dinamarca, José I Jerias, Daniel A Soto, Jorge A Soto, Natalia V Díaz, Yessica Y Leyton, Rodrigo A Villegas, Alexis M Kalergis, Abel E Vásquez
Group B Streptococcus (GBS) is the leading cause of neonatal meningitis and a common pathogen in livestock and aquaculture industries around the world. Conjugate polysaccharide and protein-based vaccines are under development. The surface immunogenic protein (SIP) is a conserved protein in all GBS serotypes and has been shown to be a good target for vaccine development. The expression of recombinant proteins in Escherichia coli cells has been shown to be useful in the development of vaccines, and the protein purification is a factor affecting their immunogenicity...
February 13, 2018: Molecular Biotechnology
Alessandra Altamirano, Andreas Naschberger, Barbara G Furnrohr, Radka Saldova, Weston B Struwe, Patrick M Jennings, Silvia Millán Martín, Suzana Malic, Immanuel Plangger, Stefan Lechner, Reina Pisano, Nicole Peretti, Bernd Linke, Mario M Aguiar, Friedrich Fresser, Andreas Ritsch, Tihana Lenac Rovis, Christina Goode, Pauline M Rudd, Klaus Scheffzek, Bernhard Rupp, Hans Dieplinger
Afamin is an 87 kDa glycoprotein with five predicted N-glycosylation sites. Afamin's glycan abundance contributes to conformational and chemical inhomogeneity presenting great challenges for molecular structure determination. For the purpose of studying the structure of afamin, various forms of recombinantly expressed human afamin (rhAFM) with different glycosylation patterns were thus created. Wild-type rhAFM and various hypo-glycosylated forms were expressed in CHO, CHO-Lec1 and HEK293T cells. Fully non-glycosylated rhAFM was obtained by transfection of point-mutated cDNA to delete all N-glycosylation sites of afamin...
February 14, 2018: Journal of Proteome Research
Vishakha A Pawar, Anil S Prajapati, Rekha C Akhani, Darshan H Patel, R B Subramanian
A thermostable keratinase designated as KBALT was purified from Bacillus altitudinis RBDV1 from a poultry farm in Gujarat, India. The molecular weight of the native KBALT (nKBALT) purified using ammonium sulfate and ion exchange and gel permeation chromatography with a 40% yield and 80-fold purification was estimated to be ~ 43 kDa. The gene for KBALT was successfully cloned, sequenced and expressed in Escherichia coli. Recombinant KBALT (rKBALT) when purified using a single step Ni-NTA His affinity chromatography achieved a yield of 38...
February 2018: 3 Biotech
Aiganym T Zhumabek, Laura S Abeuova, Nurzhan S Mukhametzhanov, Herman B Scholthof, Yerlan M Ramankulov, Shuga A Manabayeva
Plants offer a unique combination of advantages for the production of valuable recombinant proteins in a relatively short time. For instance, a variety of diagnostic tests have been developed that use recombinant antigens expressed in plants. The envelope glycoprotein gp51 encoded by Bovine leukemia virus (BLV) is one of the essential subunits for viral infectivity. It was indicated that the recombinant gp51 (rgp51) of BLV сan be used as an synthetic alternative antigen useful in the diagnosis of BLV infection in cattle...
February 1, 2018: Journal of Virological Methods
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
Vishwa D Trivedi, Tashmay S Jones, Renee P Walker
Advances in biotechnology generated wide range of microbial genome and their related protein database. Freshwater cyanobacterium Anabaena PCC7120 sensory rhodopsin, ASR in contrast to classical haloarchaeal sensory rhodopsins interacts with putative soluble transducer, ASRT. The 125 amino acid transducer exists as a soluble protein and is involved in photoreceptor binding. Recombinant DNA tools in biotechnology conventionally support the use of affinity tags for ease of protein purification and subsequent studies...
August 2017: Int J Eng Sci (Ghaziabad)
Yuan Ge, Yunhee Kang, Robert M Cassidy, Kyung-Mee Moon, Renate Lewis, Rachel O L Wong, Leonard J Foster, Ann Marie Craig
In contrast with numerous studies of glutamate receptor-associated proteins and their involvement in the modulation of excitatory synapses, much less is known about mechanisms controlling postsynaptic GABAA receptor (GABAAR) numbers. Using tandem affinity purification from tagged GABAAR γ2 subunit transgenic mice and proteomic analysis, we isolated several GABAAR-associated proteins, including Cleft lip and palate transmembrane protein 1 (Clptm1). Clptm1 interacted with all GABAAR subunits tested and promoted GABAAR trapping in the endoplasmic reticulum...
January 17, 2018: Neuron
Christopher M Ziegler, Emily A Bruce, Jamie A Kelly, Benjamin R King, Jason W Botten
We report the development of recombinant New World (Junín; JUNV) and Old World (lymphocytic choriomeningitis virus; LCMV) mammarenaviruses that encode an HA-tagged matrix protein (Z). These viruses permit the robust affinity purification of Z from infected cells or virions, as well as the detection of Z by immunofluorescent microscopy. Importantly, the HA-tagged viruses grow with wild-type kinetics in a multi-cycle growth assay. Using these viruses, we report a novel description of JUNV Z localization in infected cells, as well as the first description of colocalization between LCMV Z and the GTPase Rab5c...
February 2018: Journal of General Virology
In Sil Jeong, Sangmin Lee, Florian Bonkhofer, Jordan Tolley, Akihito Fukudome, Yukihiro Nagashima, Kimberly May, Stephan Rips, Sang Yeol Lee, Patrick Gallois, William K Russell, Hyun Suk Jung, Antje von Schaewen, Hisashi Koiwa
The oligosaccharyltransferase (OT) complex catalyzes N-glycosylation of nascent secretory polypeptides in the lumen of the endoplasmic reticulum (ER). Despite its importance, little is known about the structure and function of plant OT complexes, mainly due to lack of efficient recombinant protein-production systems suitable for studies on large plant protein complexes. Here, we purified Arabidopsis OT complexes using tandem affinity-tagged OT subunit STAUROSPORINE AND TEMPERATURE SENSITIVE3a (STT3a) expressed by an Arabidopsis protein super-expression platform...
January 31, 2018: Plant Journal: for Cell and Molecular Biology
Cher Hui Goey, David Bell, Cleo Kontoravdi
Host cell proteins (HCPs) are endogenous impurities, and their proteolytic and binding properties can compromise the integrity, and, hence, the stability and efficacy of recombinant therapeutic proteins such as monoclonal antibodies (mAbs). Nonetheless, purification of mAbs currently presents a challenge because they often co-elute with certain HCP species during the capture step of protein A affinity chromatography. A Quality-by-Design (QbD) strategy to overcome this challenge involves identifying residual HCPs and tracing their source to the harvested cell culture fluid (HCCF) and the corresponding cell culture operating parameters...
January 30, 2018: MAbs
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
Vaishali Verma, Charanpreet Kaur, Payal Grover, Amita Gupta, Vijay K Chaudhary
The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro...
2018: PloS One
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