The determination by radiochemical assay of argininosuccinase produced in an Escherichia coli system in vitro.
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ABSTRACT:
SUBMITTER: Zidwick MJ
PROVIDER: S-EPMC1153893 | biostudies-other | 1982 Dec
REPOSITORIES: biostudies-other
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Project description:ObjectiveTo elucidate the radionuclides and radiochemical impurities included in radiosynthesis processes of positron emission tomography (PET) tracers.MethodsTarget materials and PET tracers were produced using a cyclotron/synthesis system from Sumitomo Heavy Industry. Positron and ?-ray emitting radionuclides were quantified by measuring radioactivity decay and using the high-purity Ge detector, respectively. Radiochemical species in gaseous and aqueous target materials were analyzed by gas and ion chromatography, respectively.ResultsTarget materials had considerable levels of several positron emitters in addition to the positron of interest, and in the case of aqueous target materials extremely low levels of many ?-emitters. Five 11C-, 15O-, or 18F-labeled tracers produced from gaseous materials via chemical reactions had no radionuclidic impurities, whereas 18F-FDG, 18F-NaF, and 13N-NH3 produced from aqueous materials had several ?-emitters as well as impure positron emitters. 15O-Labeled CO2, O2, and CO had a radionuclidic impurity 13N-N2 (0.5-0.7 %).ConclusionsTarget materials had several positron emitters other than the positron of interest, and extremely low level ?-emitters in the case of aqueous materials. PET tracers produced from gaseous materials except for 15O-labeled gases had no impure radionuclides, whereas those derived from aqueous materials contained acceptable levels of impure positron emitters and extremely low levels of several ?-emitters.
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Project description:Comparison of Escherichia coli proteomics of different DNA sequence binding proteins and identification of heterologous expressed protein
2021-04-14 | PXD025380 | iProX
Project description:1. A synthetic 3-([(14)C]valine)-labelled tetradecapeptide renin substrate was used to measure renin concentration. Renin liberated (14)C-labelled angiotensin I, which was separated from the labelled substrate by paper chromatography. The conversion of substrate into angiotensin I was quantitated by liquid-scintillation counting of radioactivity. 2. The rate of conversion of the substrate into angiotensin I was shown to be linearly related to renin concentration and time under suitable conditions. Angiotensin generation measured in this system agrees well with that measured by bioassay. 3. It is suggested that the use of a pure substrate offers advantages that include the standardization of current units of renin measurement.
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Project description:BackgroundThe zinc homeostasis system in Escherichia coli is one of the most intensively studied prokaryotic zinc homeostasis systems. Its underlying regulatory machine consists of repression on zinc influx through ZnuABC by Zur (Zn2+ uptake regulator) and activation on zinc efflux via ZntA by ZntR (a zinc-responsive regulator). Although these transcriptional regulations seem to be well characterized, and there is an abundance of detailed in vitro experimental data available, as yet there is no mathematical model to help interpret these data. To our knowledge, the work described here is the first attempt to use a mathematical model to simulate these regulatory relations and to help explain the in vitro experimental data.ResultsWe develop a unified mathematical model consisting of 14 reactions to simulate the in vitro transcriptional response of the zinc homeostasis system in E. coli. Firstly, we simulate the in vitro Zur-DNA interaction by using two of these reactions, which are expressed as 4 ordinary differential equations (ODEs). By imposing the conservation restraints and solving the relevant steady state equations, we find that the simulated sigmoidal curve matches the corresponding experimental data. Secondly, by numerically solving the ODEs for simulating the Zur and ZntR run-off transcription experiments, and depicting the simulated concentrations of zntA and znuC transcripts as a function of free zinc concentration, we find that the simulated curves fit the corresponding in vitro experimental data. Moreover, we also perform simulations, after taking into consideration the competitive effects of ZntR with the zinc buffer, and depict the simulated concentration of zntA transcripts as a function of the total ZntR concentration, both in the presence and absence of Zn(II). The obtained simulation results are in general agreement with the corresponding experimental data.ConclusionSimulation results show that our model can quantitatively reproduce the results of several of the in vitro experiments conducted by Outten CE and her colleagues. Our model provides a detailed insight into the dynamics of the regulatory system and also provides a general framework for simulating in vitro metal-binding and transcription experiments and interpreting the relevant experimental data.
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Project description:OBJECTIVE:The aim of present study was cloning and expression of phiC31 integrase cDNA in a bacterial expression vector. Thus, an intra molecular assay vector was applied to show in vitro activity of recombinant protein. MATERIALS AND METHODS:In this experimental study, phiC31 cDNA was subcloned into a prokaryotic expression vector and transformed into E.coli Bl21 (DE3). Recombinant phiC31 integrase was purified form the bacterial cell lysates and its activity was verified by an in vitro functional assessment. RESULTS:Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the puri?ed phiC31 integrase confirmed the size of protein (70 kDa). Finally, the functionality of purified phiC31 integrase was verified. CONCLUSION:The results of this study indicated that the purified integrase has a great potential application for in vitro site-specific integration.
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Project description:Enterotoxigenic Escherichia coli (ETEC) strain H10407 is capable of invading epithelial cell lines derived from the human ileocecum and colon in vitro. Two separate chromosomally encoded invasion loci (tia and tib) have been cloned from this strain. These loci direct nonadherent and noninvasive laboratory strains of E. coli to adhere to and invade cultured human intestinal epithelial cells. The tib locus directs the synthesis of TibA, a 104-kDa outer membrane protein that is directly correlated with the adherence and invasion phenotypes. TibA is synthesized as a 100-kDa precursor (preTibA) that must be modified for biological activity. Outer membranes of recombinant E. coli expressing TibA or preTibA were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted to nitrocellulose. The presence of glycoproteins was detected by oxidization of carbohydrates with periodate and labeling with hydrazide-conjugated digoxigenin. Only TibA could be detected as a glycoprotein. Complementation experiments with tib deletion mutants of ETEC strain H10407 demonstrate that the TibA glycoprotein is expressed in H10407, that the entire tib locus is required for TibA synthesis, and that TibA is the only glycoprotein produced by H10407. Protease treatment of intact H10407 cells removes the carbohydrates on TibA, suggesting that they are surface exposed. TibA shows homology with AIDA-I from diffuse-adhering E. coli and with pertactin precursor from Bordetella pertussis. Both pertactin and AIDA-I are members of the autotransporter family of outer membrane proteins and are afimbrial adhesins that play an important role in the virulence of these organisms. Analysis of the predicted TibA amino acid sequence indicates that TibA is also an autotransporter. Analysis of the tib locus DNA sequence revealed an open reading frame with similarity to RfaQ, a glycosyltransferase. The product of this tib locus open reading frame is proposed to be responsible for TibA modification. These results suggest that TibA glycoprotein acts as an adhesin that may participate in the disease process.
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Project description:The A/H1N1/2009 influenza pandemic made evident the need for faster and higher-yield methods for the production of influenza vaccines. Platforms based on virus culture in mammalian or insect cells are currently under investigation. Alternatively, expression of fragments of the hemagglutinin (HA) protein in prokaryotic systems can potentially be the most efficacious strategy for the manufacture of large quantities of influenza vaccine in a short period of time. Despite experimental evidence on the immunogenic potential of HA protein constructs expressed in bacteria, it is still generally accepted that glycosylation should be a requirement for vaccine efficacy.We expressed the globular HA receptor binding domain, referred to here as HA(63-286)-RBD, of the influenza A/H1N1/2009 virus in Escherichia coli using a simple, robust and scalable process. The recombinant protein was refolded and purified from the insoluble fraction of the cellular lysate as a single species. Recombinant HA(63-286)-RBD appears to be properly folded, as shown by analytical ultracentrifugation and bio-recognition assays. It binds specifically to serum antibodies from influenza A/H1N1/2009 patients and was found to be immunogenic, to be capable of triggering the production of neutralizing antibodies, and to have protective activity in the ferret model.Projections based on our production/purification data indicate that this strategy could yield up to half a billion doses of vaccine per month in a medium-scale pharmaceutical production facility equipped for bacterial culture. Also, our findings demonstrate that glycosylation is not a mandatory requirement for influenza vaccine efficacy.
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Project description:Background:Grx6 is a yeast Golgi/endoplasmic reticulum protein involved in iron-sulfur binding that belongs to monothiol glutaredoxin-protein family. Grx6 has been biochemically characterized previously. Grx6 contains a conserved cysteine residue (Cys-136). Depending on the active-site sequences, Grxs can be classified to classic dithiol Grxs with a CXXC motif known as classes II and monothiol Grxs with a CXXS motif known as classes I, and Grx6 belongs to the class I with a CSYS motif. Results:Our results showed how the loop between the N-terminal and C-terminal can affect the stability. When Grx6 was incubated with FeSO4·7H2O and (NH4)2Fe(SO4)2·6H2O, a disulfide bond was formed between the cysteine 136 and glutathione, and the concentration of dimer and tetramer was increased. The results presented various levels of stability of Grx6 with mutant and deleted amino acids. We also highlighted the difference between the monomer and dimer forms of the Grx6, in addition to comparison of the Fe-S cluster positions among holo forms of poplar Grx-C1, human Grx2 and Saccharomyces cerevisiae Grx6. Conclusions:In this paper, we used a combination of spectroscopic and proteomic techniques to analyse the sequence and to determine the affected mutations and deletions in the stability of Grx6. Our results have increased the knowledge about the differences between monomer and dimer structures in cellular processes and proteins whose roles and functions depend on YCA1 in yeast.
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