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Kaufman, Randal J. "Mammalian recombinant proteins: Structure, function and immunological analysis." Current Opinion in Biotechnology 1, no. 2 (December 1990): 141–50. http://dx.doi.org/10.1016/0958-1669(90)90023-e.
Sims, Andrew H., Manda E. Gent, Karin Lanthaler, Nigel S. Dunn-Coleman, Stephen G. Oliver, and Geoffrey D. Robson. "Transcriptome Analysis of Recombinant Protein Secretion by Aspergillus nidulans and the Unfolded-Protein Response In Vivo." Applied and Environmental Microbiology 71, no. 5 (May 2005): 2737–47. http://dx.doi.org/10.1128/aem.71.5.2737-2747.2005.
ABSTRACT Filamentous fungi have a high capacity for producing large amounts of secreted proteins, a property that has been exploited for commercial production of recombinant proteins. However, the secretory pathway, which is key to the production of extracellular proteins, is rather poorly characterized in filamentous fungi compared to yeast. We report the effects of recombinant protein secretion on gene expression levels in Aspergillus nidulans by directly comparing a bovine chymosin-producing strain with its parental wild-type strain in continuous culture by using expressed sequence tag microarrays. This approach demonstrated more subtle and specific changes in gene expression than those observed when mimicking the effects of protein overproduction by using a secretion blocker. The impact of overexpressing a secreted recombinant protein more closely resembles the unfolded-protein response in vivo.
Senear, Donald F., Robert A. Mendelson, Deborah B. Stone, Linda A. Luck, Elena Rusinova, and J. B. Alexander Ross. "Quantitative Analysis of Tryptophan Analogue Incorporation in Recombinant Proteins." Analytical Biochemistry 300, no. 1 (January 2002): 77–86. http://dx.doi.org/10.1006/abio.2001.5441.
Jerlström-Hultqvist, Jon, Britta Stadelmann, Sandra Birkestedt, Ulf Hellman, and Staffan G. Svärd. "Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome." Eukaryotic Cell 11, no. 7 (May 2012): 864–73. http://dx.doi.org/10.1128/ec.00092-12.
ABSTRACTIn recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoanGiardia intestinaliscan be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use inGiardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide–glutathioneS-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinantGiardiaproteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of theG. intestinalis26S proteasome by employing the Strep II-FLAG–tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and fromGiardia, which will allow the study of specific parasite proteins and protein complexes.
Shi, Xiaohong, and Richard M. Elliott. "Generation and analysis of recombinant Bunyamwera orthobunyaviruses expressing V5 epitope-tagged L proteins." Journal of General Virology 90, no. 2 (February 2009): 297–306. http://dx.doi.org/10.1099/vir.0.007567-0.
The L protein of Bunyamwera virus (BUNV; family Bunyaviridae) is an RNA-dependent RNA polymerase, 2238 aa in length, that catalyses transcription and replication of the negative-sense, tripartite RNA genome. To learn more about the molecular interactions of the L protein and to monitor its intracellular distribution we inserted a 14 aa V5 epitope derived from parainfluenza virus type 5, against which high-affinity antibodies are available, into different regions of the protein. Insertion of the epitope at positions 1935 or 2046 resulted in recombinant L proteins that retained functionality in a minireplicon assay. Two viable recombinant viruses, rBUNL4V5 and rBUNL5V5, expressing the tagged L protein were rescued by reverse genetics, and characterized with respect to their plaque size, growth kinetics and protein synthesis profile. The recombinant viruses behaved similarly to wild-type (wt) BUNV in BHK-21 cells, but formed smaller plaques and grew to lower titres in Vero E6 cells compared with wt BUNV. Immunofluorescent staining of infected cells showed the L protein to have a punctate to reticular distribution in the cytoplasm, and cell fractionation studies indicated that the L protein was present in both soluble and microsomal fractions. Co-immunoprecipitation and confocal microscopic assays confirmed an interaction between BUNV L and N proteins. The recombinant viruses expressing tagged L protein will be highly valuable reagents for the detailed dissection of the role of the BUNV L protein in virus replication.
Grover, J., and P. J. Roughley. "The expression of functional link protein in a baculovirus system: analysis of mutants lacking the A, B and B' domains." Biochemical Journal 300, no. 2 (June 1994): 317–24. http://dx.doi.org/10.1042/bj3000317.
Functional recombinant human link protein has been produced using a baculovirus expression system. In addition to the intact link protein, three mutant forms have also been expressed. Each mutant bears a deletion equivalent to the protein encoded by one exon in the gene. These deletions represent the A domain, which is thought to be responsible for interaction with aggrecan, and the B or B' domains, which are associated with the interaction with hyaluronate. Such deletions split codons spanning exon boundaries, but maintain the reading frame of the protein and result in the correct amino acid being present at the splice junction. All the recombinant proteins appear as two components upon SDS/PAGe, though the abundance of the two forms does vary between preparations, as a result of variable substitution by N-linked oligosaccharides. The recombinant intact link protein was able to interact with both hyaluronate and aggrecan, showing that the baculovirus system is able to produce functional molecules. All of the recombinant mutant link proteins were also able to interact with hyaluronate, indicating that both the B and B' domains can function independently. The recombinant mutant link proteins were also able to interact with aggrecan, with the exception of the mutant lacking the A domain, confirming that this ability resides entirely within this domain.
Fei, Dongliang, Yaxi Guo, Qiong Fan, Ming Li, Li Sun, Mingxiao Ma, and Yijing Li. "Codon optimization, expression in Escherichia coli, and immunogenicity analysis of deformed wing virus (DWV) structural protein." PeerJ 8 (March 2020): e8750. http://dx.doi.org/10.7717/peerj.8750.
Background Deformed wing virus (DWV) is a serious threat to honey bees (Apis mellifera) and is considered a major cause of elevated losses of honey bee colonies. However, lack of information on the immunogenicity of DWV structural proteins has hindered the development of effective biocontrol drugs. Methods We optimized the VP1, VP2 and VP3 codons of DWV surface capsid protein genes on the basis of an Escherichia coli codon bias, and the optimized genes of roVP1, roVP2 and roVP3 were separately expressed in E. coli and purified. Next, the three recombinant proteins of roVP1, roVP2 and roVP3 were intramuscularly injected into BALB/c and the immunogenicity was evaluated by the levels of specific IgG and cytokines. Furthermore, anti-roVP-antisera (roVP1 or roVP2 or roVP3) from the immunized mice was incubated with DWV for injecting healthy white-eyed pupae for the viral challenge test, respectively. Results The optimized genes roVP1, roVP2 and roVP3 achieved the expression in E. coli using SDS-PAGE and Western blotting. Post-immunization, roVP2 and roVP3 exhibited higher immunogenicity than roVP1 and stimulated a stronger humoral immune response in the mice, which showed that the recombinant proteins of roVP3 and roVP2 induced a specific immune response in the mice. In the challenge test, data regarding quantitative real-time RT-PCR (qRT-PCR) from challenged pupae showed that the level of virus copies in the recombinant protein groups was significantly lower than that of the virus-only group at 96 h post-inoculation (P < 0.05). Among them, the degree of neutralization using antibodies raised to the recombinant proteins are between approximately 2-fold and 4-fold and the virus copies of the roVP3 group are the lowest in the three recombinant protein groups, which indicated that specific antibodies against recombinant proteins roVP1, roVP2 and roVP3 of DWV could neutralize DWV to reduce the virus titer in the pupae. Collectively, these results demonstrated that the surface capsid protein of DWV acted as candidates for the development of therapeutic antibodies against the virus.
Bakli, Mahfoud, Raul Pascalau, and Laura Smuleac. "Rare Codon Analysis in Rickettsia Affecting Recombinant Protein Expression in Escherichia coli." Advanced Research in Life Sciences 4, no. 1 (January 2020): 30–35. http://dx.doi.org/10.2478/arls-2020-0015.
Abstract Rickettsia species are important emerging pathogens causing rickettsial diseases, which are important cause death worldwide. The number of recombinant proteins used for diagnostic and therapeutic applications has increased dramatically, which is important in determination of protein function, structure and antigensity. Although E. coli is widely used expression system, the codon bias can hamper protein expression due to the presence of rare codons in gene sequence coding protein of interest. Using bioinformatics tools, rare codon analysis of rickettsial genes was performed and compared to not expressed proteins in both R. prowazekii and R. rickettsii. A negative correlation between frequencies of rare codons in Rickettsia and success of rickettsial protein expression was observed. This study suggested a useful tool to improve rickettsial recombinant protein expression in E. coli.
Дисертації з теми "Recombinant proteins Analysis":
Lee, Jae-Yong. "Expression, purification and interaction analysis of recombinant SRB proteins." Electronic Thesis or Diss., Imperial College London, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.407809.
Kotlarski, Nicholas. "Process-scale renaturation of recombinant proteins from inclusion bodies /." Title page, contents and summary only, 1998. http://web4.library.adelaide.edu.au/theses/09PH/09phk87.pdf.
Kepple, Kevin V. "Analysis of the binding mechanisms and cellular targets of peptide inhibitors that block site-specific recombination in vitro /." Dissertations, Academic, Connect to a 24 p. preview or request complete full text in PDF formate. Access restricted to UC campuses, 2006. http://wwwlib.umi.com/cr/ucsd/fullcit?p3208620.
Castilho, Alexandra Marina Machado Ferreira. "Molecular cytogenic analysis of recombinant chromosomes in wheat - Aegilops umbellulata lines." Electronic Thesis or Diss., University of East Anglia, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.296341.
Development of chimeric, recombinant peptides, proteins and enzymes expands the availability of protein/enzyme–based tools for cellular analysis and new assay platforms. Ideal protein reagents for cellular analysis must translocate into a variety of cells with minimum cell damage, retain stability and biological activity within the cell during analysis, and provide a reliable, measurable signal. This work focused on development, characterization and utilization of chimeric recombinant peptide, protein and enzyme reagents for cellular analysis and immunoassays. A cell-penetrating, fluorescent protein substrate (PKAS) was developed to monitor intracellular protein kinase A activity in cells without the need for cellular transfection. PKAS translocated into HeLa cells, βTC-3 cells and pancreatic islets with minimal toxicity. Upon cellular loading, glucose dependent phosphorylation of PKAS was observed in both βTC-3 and pancreatic islets via capillary zone electrophoresis. In pancreatic islets, maximal PKAS phosphorylation (83 ± 6 %) was observed at 12 mM glucose, whereas maximal PKAS phosphorylation (86 ± 4 %) in βTC-3 cells was with 3 mM glucose indicating a left-shifted glucose sensitivity. A cell-penetrating luciferase chimera (Luc-TAT) and a cell-penetrating phospholipid nanoshell entrapped luciferase (Luc-PPN) was constructed to monitor dynamic changes in intracellular ATP levels in mammalian cells. Upon cellular loading, the activity of Luc-TAT and Luc-PPN was monitored with time. Luc-TAT lost approximately 50% activity within one hour, and decreased rapidly over time. In contrast Luc-PPNs retain approximately 95% activity in 1 hour and 77% after 12 hours showing longer biological lifetime. Luc-PPNs were able to detect dynamic ATP changes in intact HeLa cells in the presence of KCN and NaN3. The bioluminescence returned to background levels within 8-10 minutes after treatment with KCN, whereas NaN₃ showed ~ 40% reduction. Two novel recombinant human parathyroid hormone (hPTH) analogs hPTHEGFP and hPTH-Cys were prepared to develop immunoassays for PTH detection in clinical samples. Initial experiments show promise for these analogs for use in CZELIF based immunoassays. The analogs present a number of distinctive advantages for clinical assays and can be used to develop several immunoassay platforms.
Larsen, Sasha Ellen Marie. "Characterization of components that increase secretion of recombinant proteins in pichia pastoris." Text, Scholarly Commons, 2011. https://scholarlycommons.pacific.edu/uop_etds/769.
Pichia pastoris is a methylotrophic yeast that is commonly used for its ability to express and secrete heterologous proteins. However, some proteins are not readily secreted in P pastoris and so adjustments in the secretion pathway must be made in order to achieve secretion. The Lin-Cereghino lab previously developed mutant strains using restriction enzyme-mediated integration that enabled P pastoris to secrete Pgalactosidase at higher levels than the wild type strain. This study focuses on characterizing the random pREMI-Z mutations in the genomic DNA and examining their secretory phenotype, in hopes of creating a super secretor strain. The ah3 mutant was specifically chosen and characterized for its ability to secrete HRP and SLPI proteins and the effect of the pREMI-Z mutation on the morphology of the cells using transmission electron microscopy. An examination into the AH3 protein yielded a comparative B-galactosidase secretion study between the ah3 mutant and ah3 mutant cells transformed with the pKANB-AH3 rescue construct. Lastly, a cell localization experiment was done to examine where the AH3 protein may be found. These experiments help to increase the current understanding of the secretion pathway in Pichia pastoris and serves as an outline of how to characterize other pREMI -Z mutant strains.
Carre, Heather Emily. "Expression and analysis of recombinant mycoplasma hyponeumoniae proteins as potential subunit vaccine candidates." Electronic Thesis or Diss., Royal Veterinary College (University of London), 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.522182.
Moore, Shona. "An analysis of the structure and function of malarial Duffy-binding-like protein domains using recombinant fusion proteins." Electronic Thesis or Diss., University of Warwick, 2016. http://wrap.warwick.ac.uk/86933/.
Duffy-binding-like domains are present in two potential malaria vaccine candidates. Located on the merozoite surface, MSPDBL1 and MSPDBL2 have been implicated in erythrocyte invasion and identified as targets of natural immunity. Merozoite DBL domains have been shown to bind the Fc region of natural IgM. This is characteristic of several PfEMP1s, and is also well documented in bacteria, viruses and other parasites, where it is thought to prevent specific binding of the more deadly IgG antibodies. We have developed a mammalian expression system to produce merozoite DBL domains as Fc fusion proteins, facilitating investigation into their adhesive properties. Fc-fusion proteins are composed of the Fc region of IgG fused to a peptide and are a rapidly expanding field of bio-engineering. They have been successful in drug delivery due to their ability to increase serum half-life of the fused protein by the interaction of the IgG Fc with the neonatal Fc receptor (FcRn). Engineering of the Fc scaffold has shown improved receptor binding, allowing cross-linking of Fc receptors for improved vaccine design. The expression of homodimeric DBL-Fc fusions is di cult, evidenced by incorrect folding and low protein yield. A flexible ,extended hinge region was designed to increase the distance between the Fc and the fused DBL domain, and improved protein folding and IgM binding. Further work may optimise this hinge region for the development of malarial vaccines, or therapeutics for IgM-mediated diseases. The structural analysis of all known IgM-binding DBL domains and residues on the merozoite DBL surface predict the involvement of helix 2a in IgM binding. This contradicts a recent homology model of the IgM-binding interaction, and suggests that the model needs revision. An improved DBL-Fc fusion could be used to identify critical binding residues located in this helix using the more focused approach of site-directed mutagenesis.
Prasad, Alpana. "Immune function and structural analysis of recombinant bovine conglutinin and human lung surfactant protein-D." Electronic Thesis or Diss., University of Oxford, 2000. http://ora.ox.ac.uk/objects/uuid:f9a5ae66-4ed0-4bdf-90eb-c873ca44147d.
Recognition of sugar moieties on the surface of microorganisms is one of the ways the body distinguishes potential pathogens from self-cells. The sugarbinding proteins, lectins, mediate this recognition role of the first line of defence against infections, preceding the antibody-mediated (adaptive) immune response. Collectins are calcium-dependent carbohydrate-binding proteins that have been implicated in innate immunity. Bovine conglutinin (BC) and lung surfactant protein-D (SP-D), belong to the family of 'collectins' which are characterised by four domains: an N-terminal cysteine-rich region, a collagenlike region linked with the carbohydrate recognition domain (CRD) via an ahelical neck region. BC and SP-D show remarkable similarity in their amino acid sequence (79% identity), function and biological characteristics. They have been shown to mediate microbial clearance either by directly binding to bacteria leading to phagocytosis or interacting with complement system components. The present study aims to elucidate the biological function of these proteins more precisely. Recombinant fragments (r) of BC and SP-D consisting of their CRDs and neck regions have been cloned in pET-21a and pMal-c2 vectors respectively, for expression in Escherichia coli. Recombinant conglutinin was expressed in BL21(DE3)pLysS and isolated by a denaturation-renaturing procedure. Binding of rBC(N/CRD) to mannan and complement component, iC3b, was assessed in real-time by BIAcore. The dissociation constants were calculated by Scatchard analysis. The carbohydrate structures present on the surface of the microorganisms play an important role in mediating the interactions with the immune cells. The recombinant molecules showed calcium-dependent binding to lipopolysaccharides (LPS) from gram-negative bacteria Pseudomonas aeruginosa, Klebsiella pnuemonia and Salmonella typhosa, which was inhibited in presence of sugars. rBC(N/CRD) also bound to whole bacteria as assessed by ELISA and retained its capacity to recognise various complement system components and the carbohydrate moieties on the surface of various pathogenic microorganisms. The recombinant protein retained its ability to bind various sugar residues, although with lower affinity than that of the native molecule. rBC(N/CRD) is able to bind and aggregate bacteria and cause agglutination of bacterial cell suspensions. A novel model has been used to describe the interactions of the collectins at the molecular level based on specificity of carbohydrate-recognition by the collectins. The pyocin mutant strain 1291 series of Neisseria gonorrhoeae has sequential deletions of the terminal sugars in their lipooligosaccharides (LOS). Conglutinin showed a preferential high affinity binding to 1291a mutant that expresses GlcNAc as the terminal hexose, in comparison to other mutants. This provides a unique system to understand the specific cell-surface interactions in relevance to a particular lectin. Further elucidation of the function of CRD and neck region at a structural level is in progress, using X-ray crystallography. Since the submission of the thesis, the structure of the monomeric CRD has been solved, which revealed a remarkable similarity to the SP-D and MBL structure. Trials are underway to get the structure of the trimeric CRDs. These studies aim to provide a better understanding of the collectinpathogen interaction at the biological and structural levels. The ultimate aim is to determine if the recombinant forms of these proteins can be used therapeutically to enhance the uptake and killing of pathogens.
Alodailah, Sattam Sonitan. "The Generation of Recombinant Zea mays Spastin and Katanin Proteins for In Vitro Analysis." Thesis or Diss., University of North Texas, 2017. https://digital.library.unt.edu/ark:/67531/metadc1062897/.
Plant microtubules play essential roles in cell processes such as cell division, cell elongation, and organelle organization. Microtubules are arranged in highly dynamic and ordered arrays, but unlike animal cells, plant cells lack centrosomes. Therefore, microtubule nucleation and organization are governed by microtubule-associated proteins, including a microtubule-severing protein, katanin. Mutant analysis and in vitro characterization has shown that the highly conserved katanin is needed for the organization of the microtubule arrays in Arabidopsis and rice as well as in a variety of animal models. Katanin is a protein complex that is part of the AAA+ family of ATPases. Katanin is composed of two subunits, katanin-p60, a catalytic subunit and katanin-p80, a regulatory subunit. Spastin is another MT-severing protein that was identified on the basis of its homology to katanin. In animal cells, spastin is also needed for microtubule organization, but its functionality has not yet been investigated in plants. To initiate an exploration of the function of katanin-p60 and spastin in Zea mays, my research goal was to generate tools for the expression and purification of maize katanin-p60 and spastin proteins in vitro. Plasmids that express katanin-p60 and spastin with N-terminal GST tags were designed and constructed via In-Fusion® cloning after traditional cloning methods were not successful. The constructs were expressed in E. coli, then the recombinant proteins were purified. To determine if the GST-tagged proteins are functional, ATPase activity and tubulin polymerization assays were performed. While both GST-katanin-p60 and GST-spastin hydrolyzed ATP indicating that the ATPase domains are functional, the results of the tubulin polymerization assays were less clear and further experimentation is necessary.
Thomas, Sandra M. Patenting of recombinant proteins: An analysis of tissue plasminogen activator (t-PA) in Europe, United States and Japan. Brighton: University of Sussex, Science Policy Research Unit, 1994.
Частини книг з теми "Recombinant proteins Analysis":
Fitchette-Lainé, Anne-Catherine, Lise-Anne Denmat, Patrice Lerouge, and Loïc Faye. "Analysis of N- and O-Glycosylation of Plant Proteins." In Recombinant Proteins from Plants, 271–90. Totowa, NJ: Humana Press, 1998. http://dx.doi.org/10.1007/978-1-60327-260-5_19.
Tong, Kit I., Masayuki Yamamoto, and Toshiyuki Tanaka. "Selective Isotope Labeling of Recombinant Proteins in Escherichia coli." In Intrinsically Disordered Protein Analysis, 439–48. New York, NY: Springer New York, 2012. http://dx.doi.org/10.1007/978-1-4614-3704-8_30.
Arakawa, Tsutomu, and John S. Philo. "Biophysical and Biochemical Analysis of Recombinant Proteins." In Pharmaceutical Biotechnology, 19–45. New York, NY: Springer New York, 2013. http://dx.doi.org/10.1007/978-1-4614-6486-0_2.
Sominskaya, Irina, and Kaspars Tars. "Immunological Methods for Analysis of Recombinant Proteins." In Basic Cloning Procedures, 135–44. Berlin, Heidelberg: Springer Berlin Heidelberg, 1998. http://dx.doi.org/10.1007/978-3-642-71965-3_7.
Tolkatchev, Dmitri, Josee Plamondon, Richard Gingras, Zhengding Su, and Feng Ni. "Recombinant Production of Intrinsically Disordered Proteins for Biophysical and Structural Characterization." In Instrumental Analysis of Intrinsically Disordered Proteins, 653–70. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2010. http://dx.doi.org/10.1002/9780470602614.ch22.
Micsonai, András, Éva Bulyáki, and József Kardos. "BeStSel: From Secondary Structure Analysis to Protein Fold Prediction by Circular Dichroism Spectroscopy." In Methods in Molecular Biology, 175–89. New York, NY: Springer US, 2020. http://dx.doi.org/10.1007/978-1-0716-0892-0_11.
Abstract Far-UV circular dichroism (CD) spectroscopy is a classical method for the study of the secondary structure of polypeptides in solution. It has been the general view that the α-helix content can be estimated accurately from the CD spectra. However, the technique was less reliable to estimate the β-sheet contents as a consequence of the structural variety of the β-sheets, which is reflected in a large spectral diversity of the CD spectra of proteins containing this secondary structure component. By taking into account the parallel or antiparallel orientation and the twist of the β-sheets, the Beta Structure Selection (BeStSel) method provides an improved β-structure determination and its performance is more accurate for any of the secondary structure types compared to previous CD spectrum analysis algorithms. Moreover, BeStSel provides extra information on the orientation and twist of the β-sheets which is sufficient for the prediction of the protein fold. The advantage of CD spectroscopy is that it is a fast and inexpensive technique with easy data processing which can be used in a wide protein concentration range and under various buffer conditions. It is especially useful when the atomic resolution structure is not available, such as the case of protein aggregates, membrane proteins or natively disordered chains, for studying conformational transitions, testing the effect of the environmental conditions on the protein structure, for verifying the correct fold of recombinant proteins in every scientific fields working on proteins from basic protein science to biotechnology and pharmaceutical industry. Here, we provide a brief step-by-step guide to record the CD spectra of proteins and their analysis with the BeStSel method.
Mattaliano, R. J., J. J. Rosa, C. Foeller, J. P. Woodard, and M. J. Bertolini. "Analysis of Recombinant Proteins — Current Trends and Practical Limits in Analytical Stringency." In Methods in Protein Sequence Analysis · 1986, 79–95. Totowa, NJ: Humana Press, 1987. http://dx.doi.org/10.1007/978-1-59259-480-1_6.
Rozkov, Aleksei, and Sven-Olof Enfors. "Analysis and Control of Proteolysis of Recombinant Proteins in Escherichia coli." In Physiological Stress Responses in Bioprocesses, 163–95. Berlin, Heidelberg: Springer Berlin Heidelberg, 2004. http://dx.doi.org/10.1007/b95567.
Canis, Kevin, Estelle Garénaux, and Jean-François Boe. "Site-Specific N-glycosylation Analysis of Recombinant Proteins by LC/MSE." In Methods in Molecular Biology, 133–54. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1241-5_10.
Kraft, Edward, Yvonne Franke, Katharine Heeringa, Stephanie Shriver, Inna Zilberleyb, Christine Kugel, Trisha Dela Vega, et al. "Semiautomated Small-Scale Purification Method for High-Throughput Expression Analysis of Recombinant Proteins." In Methods in Molecular Biology, 51–68. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9624-7_3.
Тези доповідей конференцій з теми "Recombinant proteins Analysis":
Furis, B. C., M. J. Jorgensem, M. J. Rabiet, A. B. Contor, C. L. Brown, C. B. Shoemaker, and B. Furie. "RECOGNITION SITE DIRECTING GAMMA-CARBOXYLATION RESIDES ON THE PROPEPTIDES OF FACTOR IX AND PROTRROMBIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643992.
Factor IX and prothrombin vitamin K-dependent proteins that participate in blood coagulation undergo post-translationalmodification in which glutamic acid residues in the amino terminus of the protein are converted to gamma-carboxyglutamic acid residues. This modification confers divalent metal ion binding ability upon the proteins.As a consequence of binding divalent metal ions these proteins undergoconformational changes necessary for biological function.The vitamin K-dependent proteins are synthesized with an NH2-terminal extension. The region distal to the NH2-terminus of the mature protein is a prototypic signal sequence while the proximal region is a propeptide with homology among the vitamin K-dependent proteins. The boundary between the pre and pro sequences has been established for factor IX by analysis of three naturally occurring factor IX mutants factor IX Cambridge factor IX Oxford-3 and factor IX San Dimas, in which processing is incomplete.For human factor IX the propeptide extends from residue -18 to -1. The homology among the propeptides of vitamin K-dependent proteins suggests that the propeptide may designate adjacent gamma-carboxyglutamic acids for carboxylation. To test this hypothesis alterations in sequence were introduced into the propeptide region of human factor IX cDNA by oligonucleotide directed site specific mutagenesis.Mutated genes were expressed in Chinese hamster ovary cells. Rapid and efficient isolationof the mutant proteins by immunoaffinity chromatography permitted detailed analysis of the mutants on quantities of protein easily obtainable at low expression levels. The extent of gamma-carboxylation was assessed by the ability of the mutant proteins to interact with conformation specific antibodies directed against the gamma-carboxyglutamic acid-dependent metal stabilized native structure of factor IX as well as by direct amino acid analysis. Unmodified recombinant factor IX contained, on average, 9 gamma-carboxyglutamic acid residues, as compared to 12 for plasma factor IX. About 70% of the recombinant wild type factor IX bound to the conformation specific antibodies. Deletion of the propiece or point mutations at residues -10 or -16 led to secretion of uncarboxylated factor IX unreaotive with antibodies specific for the native structure but with the NH2-terminus of mature factor IX. In order to assess the universality of these observations we have recently cloned human prothrombin cDNA and expressed the gene in the same Chinese hamster ovary cell system used for factor IX. In contrast to factor IX, at low levels ofexpressionof the prothrombin gene, the prothrombin is fully carboxylated relative to a plasma prothrombin standard.The recombinant prothrombin exhibits the same specific clotting activity as plasma derivedprothrombin and is fully native as evaluated by conformation specific antibodies. At high levels of expression the capacityof the cells to carboxylate prothrombin can be exceeded leading to secretion of under carboxylated prothrombin. However, the absolute amount of fully carboxylated prothrombin that can be produced in this system appears to be a least fivefold greater that the absolute amount of highly carboxylated factor IX that can be synthesized.The elimination of carboxylation observed upon mutation of the propiece of factor IX suggest that the propiece contains a recognition element required for carboxylation of the protein. Assignment of a functional role to the propiece of factor IX represents the first determination of function for any pro sequence. It is anticipated that extension of these studies to prothrombin will demonstrate that this recognition signal is used by all the members of this class of proteins. In order to determine if the propiece is sufficient to designate a protein for gamma-carboxylation we are currently constructing chimeric proteins incorporating the propieceof prothrombin into the cDNA of normally uncarboxylated proteins.
Ashok Kumar, A., Margaret Insley, Jay Gambee, Sharon J. Busby, and Kathleen L. Berkner. "SITE SPECIFIC MUTAGENESIS WITHIN THE GLA-DOMAIN OF HUMAN FACTOR IX." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644079.
Factor IX, a plasma protein, plays a critical role in blood coagulation. The biological activity of factor IX as well as several other plasma proteins depends on the presence of gamma-carboxy glutamic acid (Gla) residues in their amino terminal region. In vitro mutagenesis has been used to selectively replace Gla residues of factor IX with aspartic acid (Asp) residues in order to establish the contribution of individual as well as paired Gla residues to the normal functioning of the protein. These substitutions were made at positions 7, 15, 20 and 26 in human factor IX. In addition, residue number 18, a cysteine has been changed to serine in an attempt to disrupt the highly conserved disulfide bond in the gla-domain. The gla-domain mutants will be produced in mammalian cells and compared with native recombinant factor IX. A rapid immunoaffinity purification procedure, which has been used to obtain recombinant factor IX produced in the presence or absence of vitamin K, is being used to purify the mutants. Protein sequence analysis has been used to confirm complete processing and proper gamma-carboxylation of recombinant factor IX. The properties of these mutants as compared to human factor IX will be discussed.
Higgins, Deborah L., and William E. Holmes. "CHARACTERIZATION OF RECOMBINANT HUMAN TISSUE-TYPE PLASMINOGEN ACTIVATOR MISSING THE FINGER DOMAIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643842.
Site-specific mutagenesis was used to produce a mutant form of tissue-type plasminogen activator (t-PA) which was missing the first 44 amino acids. This domain has sequence homology with the type 1 regions in proteins such as fibronectin and is commonly called the finger domain. The mutant protein (des 1-44 t-PA) was expressed in Chinese Hampster Ovary cells, and was purified using chromatography on Zn-chelate sepharose and lysine-sepharose. Sequence analysis indicated that the resulting protein was homogeneous and started at amino acid 45 in the sequence of the normal protein. The two-chain forms of both des 1-44 t-PA and normal sequence t-PA exhibited similar kinetic constants with a small synthetic substrate (H-D-Isoleucyl-L- prolyl-L-arginyl-p-nitroani 1 ide). The ability of des 1-44 t-PA to activate plasminogen was decreased to 70% of the rate of normal t-PA. The rate of plasminogen activation by normal t-PA was stimulated 51-fold in the presence of fibrin, whereas with des 1-44 t-PA it was stimulated only 40-fold. Although des 1-44 t-PA bound to lysine-agarose, little (if any) binding was observed to either intact or degraded fibrin indicating that fibrin stimulation is due in part to the ability of t-PA to recognize plasminogen bound to fibrin as a preferable substrate. The mutant t-PA was capable of forming complexes in vitro with all of the inhibitors in blood which react with normal sequence t-PA. The rate of reaction with α2-macroglobulin, however, was slower with des 1-44 t-PA than with normal sequence t-PA. The similar resistance of des 1-44 t-PA and normal sequence t-PA to proteolysis and the ability to react with a battery of monoclonal antibodies suggests that the deletion did not cause perturbed folding, but rather that alterations in function of des 1-44 t-PA were due to the lack of the finger domain.
Al-Mohannadi, Anjud Khamis, Sara Deola, and Ahmed Malki. "Visualization of Factor Viii with Flow-Cytometry as a tool for Novel Gene Therapy Approach in Hemophilia A." In Qatar University Annual Research Forum & Exhibition. Qatar University Press, 2020. http://dx.doi.org/10.29117/quarfe.2020.0164.
Haemophilia A is a genetic X-linked disorder, characterized by coagulation Factor VIII (FVIII) deficiency and leading to pathological bleedings. The disease occurs at a rate of 1 in 5000 males’ births. The treatment is the administration of plasma-derived or recombinant Factor VIII, which is expensive and leads to the development of inhibitory antibodies in around 40% of patients affected by the severe form of the disease. The disease becomes for these patients as life threatening. In new approaches to treat Haemophilia include gene therapy (GT), cells corrected through genetic modifications are used to produce in Haemophilia A patients FVIII protein in a sustained manner, as long-term treatment for this disorder. The cells of choice should be persistent and equipped with themachinery for large protein assembly and secretion. So far, target cells for Haemophilia gene correction are mostly liver cells, although they are highly immunogenic and exposed to immune-mediated destruction after GT. Based on literature evidences, bone marrow transplantation can correct Haemophilia A in mice, providing evidence that Hematopoietic stem cells (HSC) or their progeny are able to produce FVIII. We chose the approach of correcting HSC with lentiviral vectors carrying the FVIII gene cassette. Whereas classically FVIII protein is visualized on adherent cells through immunohistochemistry staining, flow-cytometry (FC) literature publications are very scarce. FC analysis is an attractive method for analysing hematopoietic cells, and in general, a versatile method for protein visualization. However, large proteins as FVIII are difficult to be carefully analysed, and the method requires several steps of optimization. This joint project with Dr. Muhammad Elnaggar, aims to optimize a method to characterize large proteins as FVIII with a reliable FC staining protocol. To this aim, we used cell lines to evaluate the expression and secretion pathways of FVIII, the intracellular requirements to fold and secrete large proteins, and the toxicities of protein accumulation, in case of GT mediated protein overexpression. For this purpose, the FC experiments were performed to optimise the FC protocol for FVIII visualization, by improving blocking efficacy, antibody-labelling efficacy and to ensure accuracy and validity through qPCR and FC double staining. This FC protocol proved its validity and usefulness in visualizing and studying functionally FVIII.
Busby, S., K. Berkner, L. Halfpap, J. Gambee, and A. Kumar. "ALTERATION OF PROPTIDE SEQUENCE IMPAIRS BIOLOGICAL ACTIVITY OF HUMAN FACTOR VII." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643784.
We have investigated the effect of altering the leader sequence of human factor VII on its biological activity. Factor VII is a vitamin K-dependent blood coagulation protein whose activity depends on the presence of gamma-carboxyglutamic acid (gla) residues in its amino terminal region. Since factor VII and other vitamin K-dependent proteins exhibit structural homology in the propeptide, it has been suggested that the propeptide is involved in gamma-carboxylation. Recently, two factor IX patients were identified with point mutations which prevented the processing of the propeptide and generated a factor IX with greatly reduced biological activity (Diuguid et al., PNAS 83; 5803; Bentley et al., Cell 45: 343). To examine this question using recombinant DNA technology, we altered the sequence of the factor VII propeptide by in vitro mutagenesis of the factor VII cDNA and then expressed the altered genes in baby hamster kidney (BHK) cells. For the 60 and 38 aa leader forms of factor VII, the arg (R) at -1 was changed to ser (S), yielding the sequence HRRRS before the +1 ala. In addition, for the 60 aa leader form, a ser was inserted after the arg at -1, resulting in the sequence HRRRRS before the +1 ala. As determined by ELISA, the mutant proteins were synthesized and secreted by BHK cells at levels comparable to the wild-type forms of factor VII. Analysis by radioimmune precipitation and SDS-PAGE indicated that substitution of arg by ser at -1 prohibits processing of the factor VII propeptide, whereas, insertion of a ser after the four arg's does not. However, all three proteins have reduced biological activity by approximately 5-fold when compared to the wild-type forms with the one-stage clotting assay. All three proteins are also quantitatively precipitated by Ba citrate, indicating they are at least partially gamma-carboxylated. These results suggest that the correct sequence of the propeptide, not just cleavage of the propeptide, is necessary for generating a biologically active molecule. The effect of these sequence alterations on gamma-carboxylation will be evaluated further by analysis of the amino acid sequence and composition of the mutant proteins.
Jorgensen, M. J., MJ Rabiet, A. B. Cantor, B. Furie, C. L. Brown, C. B. Shoemaker та B. C. Furie. "VITAMIN K-DEPENDENT γ-CARBOXYLATION OF FACTOR IX REQUIRES A RECOGNITION SITE CONTAINED WITHIN THE PROPEPTIDE". У XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643564.
The vitamin K-dependent proteins, including Factor IX (FIX), are calcium-binding proteins that undergo vitamin K-dependent post-translational modification to convert amino terminal glutamic aoid residues to Gla residues. Sequence homology among the propeptides of these proteins suggests a role for this region in designating the adjacent glutamic acid-rich domain for γ-carboxylation during intraoellular processing. Mutations vere made in the propeptide (residues -1 to -18) of FIX, and the effects on γ-carboxylation were assessed. The human FIX cDNA coding sequenoe was modified using oligonucleotide-directed site-specific mutagenesis and was expressed in Chinese hamster ovary cells. The extent of γ-carboxylation of secreted FIX was determined by (1) ability to interact with conformation-specific antibodies directed against the Gla-dependent, metal-stabilized, native structure of FIX, and (2) direct Gla analysis of the alkaline hydrolysate. Using the unmodified coding sequence, 64 ± 17 % of recombinant Factor IX bound to the conformation-specific antibodies, and 9.4 ± 0.7 Gla residues were found (compared with 12 Gla in plasma FIX). When the 18-residue propeptide was deleted, secreted FIX contained no detectable native FIX antigen and no detectable Gla. Similarly, point mutations leading to substitution of Ala for Phe at residue -16 or Glu for Ala at residue -10 led to secretion of FIX containing 2% and 6% native antigen, respectively, and approximately 1-2 Gla residues. The molecular weight of each of the reoombinant FIX species, as estimated by SDS-PAGE, was identical to that of plasma FIX. NH2-terminal sequence analysis of the mutant FIX speoies yielded the NH2-terminal sequence of plasma FIX. These data indicate that the mutations made in the propeptide did not interfere with intracellular proteolytic prooessing of FIX. We conolude that the FIX propeptide participates in defining a recognition site that designates an adjacent glutamic acid-rich domain for γ-carboxylation. The association of the propeptide with the γ-carboxylation recognition site provides the first demonstration of a specific function served by a propeptide in post-translational protein processing.
Quertermous, T., J. M. Schnee, M. S. Runge, G. R. Matsueda, N. W. Hudson, J. G. Seidman, and E. Haber. "EXPRESSION OF A RECOMBINANT ANTIBODY-TARGETED THROMBOLYTIC MOLECULE." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644616.
We have recently shown that targeting tissue-type plasminogen activator (t-PA) by covalent linkage to a fibrin-specific monoclonal antibody (59D8) produces a more potent thrombolytic agent which also induces less fibrinogenolysis. A recombinant molecule encoding a t-PA-59D8 fusion protein was constructed to provide a ready source of this agent for further study, and to allow tailoring of the active moities for maximal activity. DNA sequence coding for the 59D8 heavy chain (HC) antigen combining site was cloned from a lambdaphage library by selection with a joining region probe. Gene segments coding for this cloned HC rearrangement, the amino portion of the mouse gamma 2b HC constant region, and the catalytic B chain of t-PA were joined in the pSV2-gpt expression vector. The desired coding sequence was confirmed by nucleotide sequence analysis. The construct was transfected by electroporation into 59D8 hybridoma HC loss variants. Transfectants were screened for antifibrin antibody activity. Positive clones were shown to produce mRNA which hybridized to the human t-PA gene in Northern blot analysis. Supernatants from 5 of these clones were subjected to affinity chromatography on a synthetic fibrin-like peptide-Sepharose column followed by a benzamidine-Sepharose column. Western blots of SDS polyacrylamide gels run under reducing conditions revealed binding to a 60 kd band by a monoclonal antihuman t-PA antibody, consistent with a 59D8 HC-t-PA fusion protein. Also, binding to a 25 kd band by goat anti-mouse Fab indicated the presence of 59D8 light chain. Affinity purified protein was shown to have amidolytic activity of similar potency to t-PA in a chromogenic substrate assay utilizing S-2288. Bifunctionality of the purified protein was demonstrated first by an assay which requires the protein to bind to immobilized fibrin and simultaneously exhibit activity in the S2288 assay, and second by simultaneous fibrin and iodinated anti-t-PA binding.
Giannelli, B. F. "MOLECULAR GENETICS OF HAEMOPHILIA." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643981.
Haemophilia B, an X-linked recessive disease with an incidence of 1/30,000 newborn males, is due to defects in the gene for coagulation factor IX, which is on the long am of the X chromosome at band Xq27.1. This gene consists of approximately 34 Kb and contains 8 exons which specify a mRtfc of 2803 residues coding for a protein of 415 aa preceded by a prepro signal peptide of 46 aa. Coripanson of the functional domains of the factor IX protein with the exon structure of the gene supports the exon/protein domain hypothesis of gene evolution. The factor IX gene seems to be formed by a number of functionally and evolutionally independent modules. The signal peptide and the gla (γcarboxy-glutamic) region encoded in the first three exons are homologous to those of factor X, protein C and prothrombin. Thevfourth and fifth exons which code for the connecting peptide are homologous to one another and to the epidermal growth factor, a module that has been used in the construction of a great variety of proteins including different members of the coagulation and fibrinolytic pathways. The sixth exon encodes the activation peptide region, while the catalytic region of factor IX is coded by the seventh and eighth exon. This is at variance with other serine protease genes that have different exons for the segments containing the cardinal ami no-acids of the active centre (histidine, aspartic acid and serine).Natural selection acts against detrimental mutations of the factor IX gene and at each generation a proportion of haemophilia B genes is eliminated, as a significant number of patients does not reproduce. There appears to be no selective advantage to the heterozygote and therefore haemophilia B is maintained in the population by new mutations. Consequently, a significant proportion of patients should be born to non-carrier mothers, and unrelated patients should carry different gene defects, as recently verified by detailed analysis of individual haemophilia B genes.The defects of factor IX described so far comprise both point mutations and gene deletions. The latter affect either part or the whole of the gene and are often associated with the development of antibodies against therapeutically adninistered factor IX (the inhibitor complication). Since gene deletions may result in the complete absenceof factor IX synthesis or in the production of an extremely abnormal product, it has been suggested that mutationspreventing the synthesis of a factor IX gene product capable of inducing immune tolerance to normal factor IX is important in predisposing to the inhibitor complication.Among the point mutations described so far, those affecting the signal peptide are of particular interest. Substitutions of the arginine at positions -4 and -1 cause failure of propeptide cleavage. Thus they indicate that the propeptide consists of 18 aa an(lthat lts excision is necessary for factor IX function. It appears also that the propeptide contains a signal for γcarboxylation which has been conserved during the evolution of different γcarboxylated proteins.In spite of coagulant treatment, haemophilia B is a serious disease and one for which genetic counselling is required. Paramount for this is the detection of carriers and the diagnosis ofaffected male fetuses. DNA probes derived from the cloned factor IX gene have been used for this purpose. Carrier and first or second trimester prenatal diagnoses have been done using factors IX gene markers to follow the transmission of haemophilia B genes. Six sequence variations causing restriction fragment length polymorphisms (RFLP) in the factor IX gene have been detected and used as markers for such indirect diagnoses The efficiency of the above markers is reduced by linkage disequilibrium but, nevertheless, they offer definite carrier and nremtal diagnoses in 75-80% of the relatives of familial cases of haemophilia B.The indirect detection of gene defects is of modest help in the counselling of individuals from the families of isolated patients, but new methods for the direct detection of gene mutations promise better results in such families and also the attainment of % diagnostic success in relatives of familial cases.Finally the successful expression of recombinant factor IX genes in tissue culture and transgenic mammals raises hopes of therapeutic advances.
Zhang, Y., W. McKeand, T. Yuraszeck, W. Seifert, A. Feussner, E. Santagostino, and J. Sidhu. "Population Pharmacokinetic Analysis of Recombinant Fusion Protein Linking Coagulation Factor IX with Recombinant Albumin (rIX-FP) in Adult and Pediatric Patients with Severe Hemophilia B." In 63rd Annual Meeting of the Society of Thrombosis and Haemostasis Research. Georg Thieme Verlag KG, 2019. http://dx.doi.org/10.1055/s-0039-1680223.
Cho, Myoung-Ock, Hyo Mi Chang, Yeon Gyu Yu, Hwataik Han, and Jung Kyung Kim. "Selective and Automated Detection of Airborne Asbestos Fibers Using Chrysotile-Adhesive Protein and High-Throughput Microscopy (HTM)." In ASME 2011 International Mechanical Engineering Congress and Exposition. ASMEDC, 2011. http://dx.doi.org/10.1115/imece2011-63721.
There are several methods to detect asbestos including phase contrast microscopy (PCM), polarized light microscopy, X-ray diffraction, and electron microscopy. Although the PCM method is widely used due to its simple process and relatively low cost, it is a time-consuming and laborious process that is manually performed by a human counter. We developed a high-throughput microscopy (HTM) system for automated counting of airborne asbestos fibers to automate the conventional PCM method. Our results show that automatic image acquisition by synchronization of charge-coupled device (CCD) camera with movement of stages, and image analysis using image processing software, significantly reduced time consumption and labor. In this study, we used DksA chrysotile-adhesive protein for the selective detection of asbestos. DksA, known as the protein that specifically attaches to chrysotile, was extracted from Escherichia coli through a recombinant protein technique. We tried to detect chrysotile selectively from other fibers or particles, and we developed a highly selective and automated low-cost device for automated identification and enumeration of airborne asbestos fibers based on the HTM method.