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Affinity purification recombinant protein

Yi Ma, Jieying Yu, Jinglian Lin, Shaomin Wu, Shan Li, Jufang Wang
Human epidermal growth factor (hEGF) is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe) at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO) and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H10...
2016: BioMed Research International
Elaine Darcy, Paul Leonard, Jenny Fitzgerald, Martin Danaher, Hui Ma, Richard O'Kennedy
Affinity chromatography permits the isolation of a target analyte from a complex mixture and can be utilized to purify proteins, carbohydrates, drugs, haptens, or any analyte of interest once an affinity pair is available. It involves the exploitation of specific interactions between a binding affinity pair, such as those between an antibody and its associated antigen, or between any ligand and its associated binding receptor/protein. With the discovery of protein A in 1970, and, subsequently protein G and L, immuno-affinity chromatography has grown in popularity and is now the standard methodology for the purification of antibodies which may be implemented for a selection of different applications such as immunodiagnostics...
2017: Methods in Molecular Biology
Sinéad T Loughran, Ronan T Bree, Dermot Walls
His-tagging is the most widespread and versatile strategy used to purify recombinant proteins for biochemical and structural studies. Recombinant DNA methods are first used to engineer the addition of a short tract of poly-histidine tag (His-tag) to the N-terminus or C-terminus of a target protein. The His-tag is then exploited to enable purification of the "tagged" protein by Immobilized Metal Affinity Chromatography (IMAC). Here, we describe efficient procedures for the isolation of highly purified His-tagged target proteins from an E...
2017: Methods in Molecular Biology
Mario Lebendiker, Tsafi Danieli
Maltose-Binding Protein (MBP) is one of the most popular fusion partners being used for producing recombinant proteins in bacterial cells. MBP allows the use of a simple capture affinity step on Amylose-Agarose or Dextrin-Sepharose columns, resulting in a protein that is often 70-90 % pure in a single step. In addition to protein isolation applications, MBP provides a high degree of translation, and facilitates the proper folding and solubility of the target protein. This paper describes efficient procedures for isolating highly purified MBP target proteins...
2017: Methods in Molecular Biology
Sinéad T Loughran, Dermot Walls
Protein fusion technology has had a major impact on the efficient production and purification of individual recombinant proteins. The use of genetically engineered affinity and solubility-enhancing polypeptide "tags" has increased greatly in recent years and there now exists a considerable repertoire of these that can be used to solve issues related to the expression, stability, solubility, folding, and purification of their fusion partner. In the case of large-scale proteomic studies, the development of purification procedures tailored to individual proteins is not practicable, and affinity tags have therefore become indispensable tools for structural and functional proteomic initiatives that involve the expression of many proteins in parallel...
2017: Methods in Molecular Biology
Maria C Bewley, Lisa Reinhart, Matthew S Stake, Shorena Nadaraia-Hoke, Leslie J Parent, John M Flanagan
HIV Gag, a multidomain polyprotein that orchestrates the assembly and release of the human immunodeficiency virus (HIV), is an active target of antiretroviral inhibitor development. However, highly pure, stable, recombinant Gag has been difficult to produce in quantities sufficient for biophysical studies due to its susceptibility to proteolysis by cellular proteases during purification. Stability has been improved by using a construct that omits the p6 domain (deltap6Gag). Since in vivo, p6 is crucial to the budding process and interacts with protein complexes in the ESCRT pathway, it has been difficult to study its role the context of Gag using in vitro approaches...
October 6, 2016: Protein Expression and Purification
Dongli Guan, Zhilei Chen
Proteins purified using affinity-based chromatography often exploit a recombinant affinity tag. Existing methods for the removal of the extraneous tag, needed for many applications, suffer from poor efficiency and/or high cost. Here we describe a simple, efficient, and potentially low-cost approach-split intein-mediated ultrarapid purification (SIRP)-for both the purification of the desired tagged protein from Escherichia coli lysate and removal of the tag in less than 1 h. The N- and C-fragment of a self-cleaving variant of a naturally split DnaE intein from Nostoc punctiforme are genetically fused to the N-terminus of an affinity tag and a protein of interest (POI), respectively...
2017: Methods in Molecular Biology
L Fu, Y L Hou, X Ding, Y J Du, H Q Zhu, N Zhang, W R Hou
The complementary DNA (cDNA) of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide (FTL) gene was successfully cloned using reverse transcription-polymerase chain reaction technology. We constructed a recombinant expression vector containing FTL cDNA and overexpressed it in Escherichia coli using pET28a plasmids. The expressed protein was then purified by nickel chelate affinity chromatography. The cloned cDNA fragment was 580 bp long and contained an open reading frame of 525 bp. The deduced protein sequence was composed of 175 amino acids and had an estimated molecular weight of 19...
August 30, 2016: Genetics and Molecular Research: GMR
Amballa Chaitanyakumar, M Anbalagan
Enzymes find their applications in various industries, due to their error free conversion of substrate into product. Tannase is an enzyme used by various industries for degradation of tannin. Biochemical characterization of a specific enzyme from one organism to other is one of the ways to search for enzymes with better traits for industrial applications. Here, tannase encoding gene from Staphylococcus lugdunensis was cloned and suitability of the enzyme in various conditions was analysed to find its application in various industry...
December 2016: AMB Express
Timothy M Pabst, Michaela Wendeler, Xiangyang Wang, Sandra Bezemer, Pim Hermans, Alan K Hunter
Interest in new and diverse classes of molecules such as recombinant toxins, enzymes, and blood factors continues to grow for use a biotherapeutics. Compared to monoclonal antibodies, these novel drugs typically lack a commercially available affinity chromatography option, which leads to greater process complexity, longer development timelines, and poor platformability. To date, for both monoclonal antibodies and novel molecules, affinity chromatography has been mostly reserved for separation of process-related impurities such as host cell proteins and DNA...
September 27, 2016: Biotechnology Journal
Mahdi Abbasian, Hadieh Alsadat Eslampanah Seyedi, Badraldin Ebrahim Sayed Tabatabaei, Zahra Arab-Bafrani, Mohammad Reza Mofid, Reza Zareie
Vessel dilator is a 3.9-KDa potent anticancer peptide and a valuable candidate in the treatment of conditions such as congestive heart failure and acute renal failure amongst others. Here we report the recombinant production of vessel dilator in Escherichia coli. Three different synthetic ORF's dubbed VDI, VDII and VDIII, each encoding a trimmer of the vessel dilator peptide attached to a His tag sequence at their C- terminal, were synthesized and placed in pET21c expression vectors. The highest yield, following expression in E...
January 2017: Protein Expression and Purification
Thomas K Karikari, Alexandra Turner, Robert Stass, Leonie C Y Lee, Bethany Wilson, David A Nagel, Eric J Hill, Kevin G Moffat
Recombinant tau protein is widely used to study the biochemical, cellular and pathological aspects of tauopathies, including Alzheimer's disease and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTPD-17). Pure tau in high yield is a requirement for in vitro evaluation of the protein's physiological and toxic functions. However, the preparation of recombinant tau is complicated by the protein's propensity to aggregate and form truncation products, necessitating the use of multiple, time-consuming purification methods...
September 20, 2016: Protein Expression and Purification
Hui Chin Goh, Radoslaw M Sobota, Farid J Ghadessy, Saurabh Nirantar
Protein purification typically involves expressing a recombinant gene comprising a target protein fused to a suitable affinity tag. After purification, it is often desirable to remove the affinity tag to prevent interference with downstream functions of the target protein. This is mainly accomplished by placing a protease site between the tag and the target protein. Typically, a small oligopeptide 'stub' C-terminal to the cleavage site remains attached to the target protein due to the requirements of sequence-specific proteases...
January 2017: Protein Expression and Purification
Elvin D de Araujo, Mulu Geletu, Patrick T Gunning
STAT5B, a ubiquitious transcription factor, has been implicated in the onset and progression of several cancers. Since the inhibition of STAT activity holds significant therapeutic potential, there is a need to develop high-throughput biophysical screening platforms to rapidly identify high affinity binders of STATs. Biophysical assays would benefit from the efficient and cost-effective production of high purity, full-length STAT proteins. Herein, we have sampled a large region of protein expression and purification space that has substantially increased recombinant STAT5B protein yields from Escherichia coli...
January 2017: Protein Expression and Purification
Alexander Falkenhagen, Sabah Asad, Stanley E Read, Sadhna Joshi
BACKGROUND: Recombinant proteins of therapeutic use are ideally produced in human cells to ensure appropriate co- and post-translational modifications. However, purification of secreted proteins from the culture media is impeded by low expression from transfected cell lines and the presence of serum proteins. Here we describe a simple and cost-effective approach based on lentiviral vector-mediated gene delivery and expression of a secreted His-tagged protein from human embryonic kidney 293 T cells and direct affinity chromatography purification from the cell culture media...
2016: BMC Biotechnology
Timothy A Vickers, Stanley T Crooke
Protein-nucleic acid interactions play a crucial role in the regulation of diverse biological processes. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination, and RNA processing continues to be a crucial aspect of understanding of cell biology and the mechanisms of disease. In addition, proteins have been demonstrated to interact with antisense oligonucleotide therapeutics in a sequence and chemistry dependent manner, influencing ASO potency and distribution in cells and in vivo...
2016: PloS One
Daichao Wu, Lingzhi Qu, Yang Fu, Jun Li, Longying Jiang, Xiaojuan Chen, Ming Guo, Zhuchu Chen, Lin Chen, Yongheng Chen
PTEN-induced putative kinase 1 (PINK1) is a Ser/Thr kinase that specifically localizes on the mitochondrial membrane. It cooperates with Parkin to regulate mitochondrial quality control. Mutations in PINK1 protein which account for 8-15% of Parkinson's disease (PD), are the second most common cause of early-onset Autosomal Recessive Parkinson's disease (AR-PD). The lack of methods for PINK1 heterologous expression and purification has slowed progress in the AR-PD research field. To pave the way for direct structural study of this important protein, in this study, we developed an efficient expression system of recombinant PINK1 kinase domain (rPINK1) using Pichia pastoris (P...
December 2016: Protein Expression and Purification
Deeba Amraiz, Najam-Us-Sahar Sadaf Zaidi, Munazza Fatima
NS5 is the largest and most conserved protein among the four dengue virus (DENV) serotypes. It has been the target of interest for antiviral drug development due to its major role in replication. NS5 consists of two domains, the N-terminal methyltransferase domain and C-terminal catalytic RNA-dependent RNA polymerase (RdRp) domain. It is an unstable protein and is prone to inactivation upon prolonged incubation at room temperature, thus affecting the inhibitor screening assays. In the current study, we expressed and purified DENV RdRp alone in Esherichia coli (E...
October 2016: Enzyme and Microbial Technology
Gesa Gieseler, Iliyana Pepelanova, Lena Stuckenberg, Louis Villain, Volker Nölle, Uwe Odenthal, Sascha Beutel, Ursula Rinas, Thomas Scheper
In this study, we present the development of a process for the purification of recombinant human bone morphogenetic protein-2 (rhBMP-2) using mixed-mode membrane chromatography. RhBMP-2 was produced as inclusion bodies in Escherichia coli. In vitro refolding using rapid dilution was carried out according to a previously established protocol. Different membrane chromatography phases were analyzed for their ability to purify BMP-2. A membrane phase with salt-tolerant properties resulting from mixed-mode ligand chemistry was able to selectively purify BMP-2 dimer from refolding mixtures...
August 20, 2016: Applied Microbiology and Biotechnology
Rozálie Hexnerová, Květoslava Křížková, Milan Fábry, Irena Sieglová, Kateřina Kedrová, Michaela Collinsová, Pavlína Ullrichová, Pavel Srb, Christopher Williams, Matthew P Crump, Zdeněk Tošner, Jiří Jiráček, Václav Veverka, Lenka Žáková
Insulin and insulin-like growth factors I and II are closely related protein hormones. Their distinct evolution has resulted in different yet overlapping biological functions with insulin becoming a key regulator of metabolism, whereas insulin-like growth factors (IGF)-I/II are major growth factors. Insulin and IGFs cross-bind with different affinities to closely related insulin receptor isoforms A and B (IR-A and IR-B) and insulin-like growth factor type I receptor (IGF-1R). Identification of structural determinants in IGFs and insulin that trigger their specific signaling pathways is of increasing importance in designing receptor-specific analogs with potential therapeutic applications...
September 30, 2016: Journal of Biological Chemistry
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