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Protein expression and purification

Ji-Hua Yu, Yang-Yang Li, Mian Xiang, Jian-Quan Zhu, Xin-He Huang, Wan-Jun Wang, Rui Tan, Jia-Yu Zhou, Hai Liao
OBJECTIVES: To clone and characterize a novel bi-functional α-amylase/subtilisin inhibitor (LASI) from the rhizome of Ligusticum chuanxiong, a traditional Chinese medicine. RESULTS: The LASI showed strong homology with members of the Kunitz trypsin inhibitor family. Its putative amino acid sequence has a 40 % identity with that of the α-amylase/subtilisin inhibitor from rice. LASI gene without signal peptide was expressed in E. coli Rosetta. After purification, the recombinant LASI protein was inhibitory against not only α-amylase from porcine pancreas, Helicoverpa armigera, Spodoptera litura and Plutella xylostella, but also subtilisin A, but not against trypsin or chymotrypsin...
October 17, 2016: Biotechnology Letters
Maurizio Cirilli, Oxana Bereshchenko, Olga Ermakova, Claus Nerlov
BACKGROUND: C/EBPa and C/EBPb are transcription factors with tissue specific expression regulating several important cellular processes. They work by recruiting protein complexes to a common DNA recognition motif and both are able to compensate each other's absence in many cell types, thus showing functional redundancy. They also play distinct roles in specific cellular pathways and their abnormal functioning gives raise to different human pathologies. METHODS: To investigate the molecular basis of C/EBPa and C/EBPb specificity and redundancy we characterized their in vivo protein-protein interaction networks by Tandem Affinity Purification and Mass Spectrometry...
October 13, 2016: Biochimica et Biophysica Acta
Shalom D Goldberg, Rosa M F Cardoso, Tricia Lin, Tracy Spinka-Doms, Donna Klein, Steven A Jacobs, Vadim Dudkin, Gary Gilliland, Karyn T O'Neil
Targeted delivery of therapeutic payloads to specific tissues and cell types is an important component of modern pharmaceutical development. Antibodies or other scaffold proteins can provide the cellular address for delivering a covalently linked therapeutic via specific binding to cell-surface receptors. Optimization of the conjugation site on the targeting protein, linker chemistry and intracellular trafficking pathways can all influence the efficiency of delivery and potency of the drug candidate. In this study, we describe a comprehensive engineering experiment for an EGFR binding Centyrin, a highly stable fibronectin type III (FN3) domain, wherein all possible single-cysteine replacements were evaluated for expression, purification, conjugation efficiency, retention of target binding, biophysical properties and delivery of a cytotoxic small molecule payload...
October 13, 2016: Protein Engineering, Design & Selection: PEDS
Xiuxia Liu, Wei Zhang, Zihao Zhao, Xiaofeng Dai, Yankun Yang, Zhonghu Bai
Corynebacterium glutamicum, a Gram-positive bacterium, has been widely used for the industrial production of amino acids, such as glutamate and lysine, for decades. Due to several characteristics - its ability to secrete properly folded and functional target proteins into culture broth, its low levels of endogenous extracellular proteins and its lack of detectable extracellular hydrolytic enzyme activity - C. glutamicum is also a very favorable host cell for the secretory production of heterologous proteins, important enzymes, and pharmaceutical proteins...
October 13, 2016: Critical Reviews in Biotechnology
René Heppner, Nicola Weichert, Angelika Schierhorn, Udo Conrad, Markus Pietzsch
Spider dragline is used by many members of the Araneae family not only as a proteinogenic safety thread but also for web construction. Spider dragline has been shown to possess high tensile strength in combination with elastic behavior. This high tensile strength can be attributed to the presence of antiparallel β-sheets within the thread; these antiparallel β-sheets are why the protein is classified as a silk. Due to the properties of spider silk and its technical and medical uses, including its use as a suture material and as a scaffold for tissue regeneration, spider dragline is a focus of the biotechnology industry...
October 9, 2016: International Journal of Molecular Sciences
Darkhan Utepbergenov, Paulina M Hennig, Urszula Derewenda, Mykhaylo V Artamonov, Avril V Somlyo, Zygmunt S Derewenda
Ribosomal S6 kinases (RSK) play important roles in cell signaling through the mitogen-activated protein kinase (MAPK) pathway. Each of the four RSK isoforms (RSK1-4) is a single polypeptide chain containing two kinase domains connected by a linker sequence with regulatory phosphorylation sites. Here, we demonstrate that full-length RSK2-which is implicated in several types of cancer, and which is linked to the genetic Coffin-Lowry syndrome-can be overexpressed with high yields in Escherichia coli as a fusion with maltose binding protein (MBP), and can be purified to homogeneity after proteolytic removal of MBP by affinity and size-exclusion chromatography...
2016: PloS One
Adam J Carroll
Translation of mitochondrial encoded mRNAs by mitochondrial ribosomes is thought to play a major role in regulating the expression of mitochondrial proteins. However, the structure and function of plant mitochondrial ribosomes remains poorly understood. To study mitochondrial ribosomes, it is necessary to separate them from plastidic and cytosolic ribosomes that are generally present at much higher concentrations. Here, a straight forward protocol for the preparation of fractions highly enriched in mitochondrial ribosomes from plant cells is described...
2017: Methods in Molecular Biology
Barry J Ryan, Gemma K Kinsella
Differential protein precipitation is a rapid and economical step in protein purification and is based on exploiting the inherent physicochemical properties of the polypeptide. Precipitation of recombinant proteins, lysed from the host cell, is commonly used to concentrate the protein of choice before further polishing steps with more selective purification columns (e.g., His-Tag, Size Exclusion, etc.). Recombinant proteins can also precipitate naturally as inclusion bodies due to various influences during overexpression in the host cell...
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
Régine Brielle, Marie-Laure Pinel-Marie, Sophie Chat, Reynald Gillet, Brice Felden
Polysomes are macromolecular complexes made up of multiple ribosomes simultaneously translating a single mRNA into polypeptide chains. Together, the cellular mRNAs translated in this way are referred to 'translatome.' Translation determines a cell's overall gene expression profile. Studying translatome leads to a better understanding of the translational machinery and of its complex regulatory pathways. Given its fundamental role in cell homeostasis and division, bacterial translation is an important target for antibiotics...
October 8, 2016: Methods: a Companion to Methods in Enzymology
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
Bijan Soleymani, Sayed Hassan Hafezian, Ghodratollah Rahimi Mianji, Kamran Mansouri, Broomand Chaharaein, Ahmad Tajehmiri, Mehdi Sharifi Tabar, Ali Mostafaie
Sex determining region Y gene (SRY) is located on Y chromosome and encodes a protein with 229 amino acids. In this study, ORF region of SRY with a length of 690 bp was synthesized using PCR and ligated to pET28a (+), then transformed in E.coli DH5α. E.coli BL21 (DE3) strain was chosen to express recombinant bovine SRY protein. A set of optimization steps was taken including different concentrations of IPTG, glucose, and temperatures at differed incubation times after the induction. Results showed that temperature points and different concentrations of IPTG and glucose had a significant effect (p < 0...
August 10, 2016: Animal Biotechnology
Raghav Joshi, Edi Goihberg, Wenying Ren, Monika Pilichowska, Paul Mathew
: The haematopoietic niche is contributed to by bone marrow-resident mesenchymal stromal cells (BM-MSCs) and subverted by prostate cancer cells. To study mechanisms by which BM-MSCs and prostate cancer cells may interact, we assessed the migration, invasion, adhesion and proliferation of bone-derived prostate cancer cells (PC-3) in co-culture with pluripotent human BM-MSCs. We observed a strong adhesive, migratory and invasive phenotype of PC-3 cells with BM- MSC-co-culture and set out to isolate and characterize the bioactive principle...
August 11, 2016: Cell Adhesion & Migration
Changhua Shi, Tzu-Chiang Han, David W Wood
Fusions of elastin-like peptide (ELP) purification tags and self-cleaving inteins provide a powerful platform for purifying tagless recombinant proteins without the need for conventional packed-bed columns. A drawback to this method has been premature cleaving of the ELP tag during expression, before the purification procedure can take place. Here we demonstrate a split-intein method, where the self-cleaving intein is divided into two inactive segments during expression and purification. Spontaneous assembly of the purified intein segments then restores self-cleaving activity to deliver the tagless target protein...
2017: Methods in Molecular Biology
Andrew Scott, Marek J Noga, Paul de Graaf, Ilja Westerlaken, Esengul Yildirim, Christophe Danelon
The goal of bottom-up synthetic biology culminates in the assembly of an entire cell from separate biological building blocks. One major challenge resides in the in vitro production and implementation of complex genetic and metabolic pathways that can support essential cellular functions. Here, we show that phospholipid biosynthesis, a multiple-step process involved in cell membrane homeostasis, can be reconstituted starting from the genes encoding for all necessary proteins. A total of eight E. coli enzymes for acyl transfer and headgroup modifications were produced in a cell-free gene expression system and were co-translationally reconstituted in liposomes...
2016: PloS One
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
Lauri Reuter, Anneli Ritala, Markus Linder, Jussi Joensuu
Hydrophobin fusion technology has been applied in the expression of several recombinant proteins in plants. Until now, the technology has relied exclusively on the Trichoderma reesei hydrophobin HFBI. We screened eight novel hydrophobin tags, T. reesei HFBII, HFBIII, HFBIV, HFBV, HFBVI and Fusarium verticillioides derived HYD3, HYD4 and HYD5, for production of fusion proteins in plants and purification by two-phase separation. To study the properties of the hydrophobins, we used N-terminal and C-terminal GFP as a fusion partner...
2016: PloS One
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
Roy Baas, Ayestha Sijm, Hetty A A M van Teeffelen, Robert van Es, Harmjan R Vos, H Th Marc Timmers
Gene-specific transcription factors (GSTFs) control gene transcription by DNA binding and specific protein complex recruitment, which regulates promoter accessibility for transcription initiation by RNA polymerase II. Mutations in the GSTFs Suppressor of Mothers Against Decapentaplegic 2 (SMAD2) and SMAD4 are frequently associated with colon and rectal carcinomas. These proteins play an important role in bone morphogenic protein (BMP) and transforming growth factor β (TGF-β) signaling pathways controlling cell fate and proliferation...
October 4, 2016: Journal of Biological Chemistry
Khadijeh Alishah, Sedigheh Asad, Khosro Khajeh, Neda Akbari
Uric acid, a side product of nucleotide metabolism, should be cleared from blood stream since its accumulation can cause cardiovascular diseases and gout. Uricase (urate oxidase) converts uric acid to 5-hydroxyisourate, but it is absent in human and other higher apes. Yet, the recombinant form of uricase, Rasburicase, is now commercially available to cure tumor lysis syndrome by lowering serum uric acid level. Developing new methods to efficiently purify pharmaceutical proteins like uricase has attracted researchers' attention...
November 2016: Enzyme and Microbial Technology
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