Removal Of Nucleic Acids Research Articles

Impact of incomplete DNase I treatment on human macrophage proteome analysis

The aim of our study was to analyze the proteomic pattern of human macrophages obtained over a 4 year period from blood donors. The purpose was to simulate a long-term clinical study to assess the application of 2-D DIGE technique for differential proteomic analysis of these scarce samples. Bioinformatic analysis of 2-D DIGE gels of 19 different cultures of macrophages assessed whether they did or did not contain at least specific five spots identified by MS as being or containing bovine deoxyribonuclease I (DNase I). Bovine DNase I was used during sample treatment to remove nucleic acids from protein extracts. Macrophages were classified in two groups, which appeared to be differentiated by the completeness of DNase I treatment. Further detailed analysis revealed a different proteomic pattern of macrophage protein samples according to the completeness of this treatment. The major group of proteins affected, accounting for one third of the differentially expressed proteins, included proteins involved in cell motion and actin cytoskeleton reorganization. The use of DNase I for the removal of nucleic acids from protein samples must be avoided in proteomic studies since it can generate bias in the analysis of protein expression patterns.

Protein carbonylation: avoiding pitfalls in the 2,4-dinitrophenylhydrazine assay

Protein carbonyl content is widely used as both a marker for oxidative stress and a measure of oxidative damage. Widely used methods for determination of protein carbonylation utilize the reaction of carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH) to form protein-bound 2,4-dinitrophenylhydrazones. Hydrazones can be quantitated spectrophotometrically or, for greater sensitivity, detected immunochemically with anti-dinitrophenyl antibodies. Attention to methodology is important to avoid artifactual elevation in protein carbonyl measurements. We studied extracts of Escherichia coli to identify and eliminate such effects. Nucleic acid contamination caused serious artifactual increases in the protein carbonyl content determined by spectrophotometric techniques. Both in vitro synthesized DNA oligonucleotides and purified chromosomal DNA reacted strongly with 2,4-DNPH. Treatment of cell extracts with DNase+RNase or with streptomycin sulfate to precipitate nucleic acids dramatically reduced the apparent carbonyl, while exposure to proteinase K did not. The commercial kit for immunochemical detection of protein carbonylation (OxyBlot™ from Chemicon/Millipore) recommends a high concentration of thiol in the homogenizing buffer. We found this recommendation leads to an artifactual doubling of the protein carbonyl, perhaps due to a thiol-stimulated Fenton reaction. Avoiding oxidizing conditions, removal of nucleic acids, and prompt assay of samples can prevent artifactual effects on protein carbonyl measurements.

Role of Conserved Salt Bridges in Homeodomain Stability and DNA Binding

The sequence information available for homeodomains reveals that salt bridges connecting pairs 19/30, 31/42, and 17/52 are frequent, whereas aliphatic residues at these sites are rare and mainly restricted to proteins from homeotherms. We have analyzed the influence of salt and hydrophobic bridges at these sites on the stability and DNA binding properties of human Hesx-1 homeodomain. Regarding the protein stability, our analysis shows that hydrophobic side chains are clearly preferred at positions 19/30 and 31/42. This stabilizing influence results from the more favorable packing of the aliphatic side chains with the protein core, as illustrated by the three-dimensional solution structure of a thermostable variant, herein reported. In contrast only polar side chains seem to be tolerated at positions 17/52. Interestingly, despite the significant influence of pairs 19/30 and 31/42 on the stability of the homeodomain, their effect on DNA binding ranges from modest to negligible. The observed lack of correlation between binding strength and conformational stability in the analyzed variants suggests that salt/hydrophobic bridges at these specific positions might have been employed by evolution to independently modulate both properties.

Nucleic Acids in Protein Samples Interfere with Phosphopeptide Identification by Immobilized-Metal-Ion Affinity Chromatography and Mass Spectrometry

Immobilized-metal-ion affinity chromatography (IMAC) is used extensively for phosphopeptide enrichment in phosphoproteomics. However, the effect of nucleic acids in protein samples on phosphopeptide enrichment by IMAC has not yet been well clarified. In this study, we demonstrate that IMAC beads possess a strong adsorption of nucleic acids, especially single-stranded or single-stranded-region-containing nucleic acids, leading to approximately 50% loss of phosphopeptides during the process of IMAC enrichment. Therefore, nucleic acids must be removed from protein samples prior to IMAC. Acetonitrile (ACN) precipitation, a simple and efficient procedure, was established to remove nucleic acids from the protein samples. We showed that ACN precipitation approximately doubled the phosphopeptide number identified by IMAC and mass spectrometry, indicating that nucleic acid removal significantly improves the identification of phosphopeptides.

Process development for production of human granulocyte-colony stimulating factor by high cell density cultivation of recombinant Escherichia coli

The fed-batch process using glucose as the sole source of carbon and energy with exponential feeding rate was carried out for high cell density cultivation of recombinant Escherichia coli BL21 (DE3) expressing human granulocyte-colony stimulating factor (hG-CSF). IPTG was used to induce the expression of hG-CSF at 48 g dry cell wt l(-1) during high cell density culture of recombinant E. coli BL21 (DE3) [pET23a-g-csf]. The final cell density, specific yield and overall productivity of hG-CSF were obtained as approximately 64 g dry cell wt l(-1), 223 mg hG-CSF g(-1) dry cell wt and 775 mg hG-CSF l(-1) h(-1), respectively. The resulting purification process used cell lysis, inclusion body (IB) preparation, refolding, DEAE and Butyl-Sepharose. Effects of different process conditions such as cell lysis and washing of IB were evaluated. The results reveal that the cells lyzed at 1,200 bar, 99.9% and Triton removed about 64% of the LPS but sarcosyl had no effect on removal of nucleic acids and LPS. Further analysis show that DEAE column removes DNA about 84%. Cupper concentration was identified as parameter that could have a significant impact on aggregation, as an unacceptable pharmaceutical form that decrease process yields. The purity of purified hG-CSF was more than 99%. Also the comparison of activity between purified hG-CSF and commercial form do not show valuable decrease in activity in purified form.

The Influence of Sample Preparation and Replicate Analyses on HeLa Cell Phosphoproteome Coverage

Ongoing optimization of proteomic methodologies seeks to improve both the coverage and confidence of protein identifications. The optimization of sample preparation, inclusion of technical replicates (repeated instrumental analysis of the same sample), and biological replicates (multiple individual samples) are crucial in proteomic studies to avoid the pitfalls associated with single point analysis and under-sampling. Phosphopeptides were isolated from HeLa cells and analyzed by nano-reversed phase liquid chromatography electrospray ionization tandem mass spectrometry (nano-RP-LC-MS/MS). We observed that a detergent-based protein extraction approach, followed with additional steps for nucleic acid removal, provided a simple alternative to the broadly used Trizol extraction. The evaluation of four technical replicates demonstrated measurement reproducibility with low percent variance in peptide responses at approximately 3%, where additional peptide identifications were made with each added technical replicate. The inclusion of six technical replicates for moderately complex protein extracts (approximately 4000 uniquely identified peptides per data set) affords the optimal collection of peptide information.

Hydrophilic Residues 526KNDAAD531 in the Flexible C-terminal Region of the Chaperonin GroEL Are Critical for Substrate Protein Folding within the Central Cavity

The final 23 residues in the C-terminal region of Escherichia coli GroEL are invisible in crystallographic analyses due to high flexibility. To probe the functional role of these residues in the chaperonin mechanism, we generated and characterized C-terminal truncated, double ring, and single ring mutants of GroEL. The ability to assist the refolding of substrate proteins rhodanese and malate dehydrogenase decreased suddenly when 23 amino acids were truncated, indicating that a sudden change in the environment within the central cavity had occurred. From further experiments and analyses of the hydropathy of the C-terminal region, we focused on the hydrophilicity of the sequence region (26 KNDAAD 531 and generated two GroEL mutants where these residues were changed to a neutral hydropathy sequence (526 GGGAAG 531) and a hydrophobic sequence (526 IGIAAI 531), respectively. Very interestingly, the two mutants were found to be defective in function both in vitro and in vivo. Deterioration of function was not observed in mutants where this region was replaced by a scrambled (526 NKADDA 531) or homologous (526 RQEGGE 531) sequence, indicating that the hydrophilicity of this sequence was important. These results highlight the importance of the hydrophilic nature of 526 KNDAAD 531 residues in the flexible C-terminal region for proper protein folding within the central cavity of GroEL.

Improving purification of recombinant human interferon γ expressed in Escherichia coli; effect of removal of impurity on the process yield

Process development and optimization studies were performed in order to improve the purification process of (rhIFN-γ). The objective was to generate material with higher purity and quantity. An in-process control screening was developed to obtain the optimal condition for column chromatographic purification by measuring LPS, nucleic acids, rhIFN- γ, monomer and its covalent dimers. A new resin screening method was applied to select optimal resin for each of the chromatographic columns. The resulting process used Butyl and Q-Sepharose, refolding and SP-Sepharose for purification of IFN-γ. Effects of different process conditions such as cell lysis, removal of impurity and oxygen concentration were evaluated. Removal of impurities was evaluated by washing of inclusion bodies with 1% Triton X-100 and 3 M urea and different chromatography steps. The results reveal that Triton removed about 43% of the LPS but urea had no effect on removal of nucleic acids and LPS. Further analysis show that removal of impurities by column chromatography decreases aggregation and increases the process yield. Oxygen concentration was identified as parameter that could have a significant impact on covalent dimers formation, as an unacceptable pharmaceutical form of rhIFN-γ. On the basis of small-scale studies, optimum operating conditions were chosen and the purification process was successfully scaled-up to a pilot scale process with step yield and product quality that were better than previous reports.

ATR-IR spectroscopy as applied to nucleic acid films

For the first time the ATR technique was applied to obtain IR absorption spectra of DNA and RNA dry films. There was worked out procedure of the nucleic acid removal from germanium plate, which obviously was a main obstacle to application of ATR-IR spectroscopy to nucleic acids. This technique of IR spectroscopy was applied to confirmation of RNA tropism of aurin tricarboxylic acid observed by molecular biological methods.

Basophilia, acidophilia and argyrophilia of “dark” (compacted) neurons during their formation, recovery or death in an otherwise undamaged environment

By means of a condenser discharge electric shock paradigm, “dark” (compacted) granule neurons were momentarily produced in the hippocampal dentate gyri of rats, which were sacrificed either immediately or following survival periods ranging from 1 h to 30 days. Except for the morphological changes related to the formation, recovery or death of the “dark” neurons, the affected brain areas remained undamaged. Vibratome, frozen, cryostat and paraffin sections were stained with Mayer's hematoxylin, acid fuchsin, or Fluoro-Jade and by three silver methods widely used for the demonstration of damaged neurons; with or without previous removal of nucleic acids, partial digestion of proteins or blockade of the negatively charged side-groups of nucleic acids and proteins. The results allowed the following conclusions: (i) “Dark” neurons acquire argyrophilia and excess basophilia simultaneously with their momentary formation. (ii) Negatively charged protein molecules are responsible for these processes. (iii) From the recovering “dark” neurons, the acquired basophilia and argyrophilia disappear within a few hours post-insult. (iv) From the moribund or dead “dark” neurons, the acquired basophilia disappears in the same period of time while the acquired argyrophilia in a few days. (vi) Freshly-produced or recovering “dark” neurons are slightly acidophilic, whereas the moribund or dead ones display intense acidophilia.

Isolation of intact aortic valve scaffolds for heart-valve bioprostheses: extracellular matrix structure, prevention from calcification, and cell repopulation features.

Extracellular matrix (ECM) scaffolds isolated from valvulated conduits can be useful in developing durable bioprostheses by tissue engineering provided that anatomical shape, architecture, and mechanical properties are preserved. As evidenced by SEM, intact scaffolds were derived from porcine aortic valves by the combined use of Triton X-100 and cholate (TRI-COL) or N-cetylpyridinium (CPC) and subsequent nucleic acid removal by nuclease. Both treatments were effective in removing most cells and all the cytomembranes, with preservation of (1) endothelium basal membranes, (2) ECM texture, including the D-periodical interaction of small proteoglycans with normally D-banded collagen fibrils, and (3) mechanical properties of the treated valves. Ultrastructural features agreed with DNA, hexosamine, and uronic acid biochemical estimations. Calcification potential, assessed by a 6-week rat subdermal model, was significantly reduced by TRI-COL/nuclease treatment. This was not true for CPC only, despite better proteoglycan preservation, suggesting that nucleic acids also are involved in calcification onset. Human fibroblasts, used to repopulate TRI-COL samples, formed mono- or multilayers on surfaces, and groups of cells also were scattered within the valve leaflet framework. A biocompatible scaffolds of this kind holds promise for production of durable valve bioprostheses that will be able to undergo probable turnover and/or remodeling by repopulating recipient cells.

Value-added food:: Single cell protein

The alarming rate of population growth has increased the demand for food production in third-world countries leading to a yawning gap in demand and supply. This has led to an increase in the number of hungry and chronically malnourished people. This situation has created a demand for the formulation of innovative and alternative proteinaceous food sources. Single cell protein (SCP) production is a major step in this direction. SCP is the protein extracted from cultivated microbial biomass. It can be used for protein supplementation of a staple diet by replacing costly conventional sources like soymeal and fishmeal to alleviate the problem of protein scarcity. Moreover, bioconversion of agricultural and industrial wastes to protein-rich food and fodder stocks has an additional benefit of making the final product cheaper. This would also offset the negative cost value of wastes used as substrate to yield SCP. Further, it would make food production less dependent upon land and relieve the pressure on agriculture. This article reviews diversified aspects of SCP as an alternative protein-supplementing source. Various potential strains and substrates that could be utilized for SCP production are described. Nutritive value and removal of nucleic acids and toxins from SCP as a protein-supplementing source are discussed. New processes need to be exploited to improve yield. In that direction the solid state fermentation (SSF) method and its advantages for SCP production are highlighted.

Nucleic acid removal from Taq polymerase preparations using an aqueous/organic biphasic system.

BioTechniquesVol. 27, No. 3 BenchmarksOpen AccessNucleic Acid Removal from Taq Polymerase Preparations Using an Aqueous/Organic Biphasic SystemAriel LouwrierAriel Louwrier*Address correspondence to Dr. Ariel Louwrier, Advanced Biotechnologies, Unit B1-B2, Longmead Business Centre, Blenheim Road, Epsom, Surrey KT19 9QQ, UK. Internet: E-mail Address: ariell@adbio.co.ukAdvanced Biotechnologies, Epsom, Surrey, UKSearch for more papers by this authorPublished Online:22 Aug 2018https://doi.org/10.2144/99273bm09AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail "Nucleic Acid Removal from Taq Polymerase Preparations Using an Aqueous/Organic Biphasic System." , 27(3), pp. 444–445FiguresReferencesRelatedDetailsCited ByA simple and efficient method for extraction of Taq DNA polymeraseElectronic Journal of Biotechnology, Vol. 18, No. 5METHODS: An alternate method for purification of recombinant Taq DNA polymerase using an aqueous two-phase systemIndustrial Biotechnology, Vol. 6, No. 5Thermally reversible inactivation of Taq polymerase in an organic solvent for application in hot start PCREnzyme and Microbial Technology, Vol. 36, No. 7Application of Optical Trapping for Cells Grown on Plates: Optimization of PCR and Fidelity of DNA Sequencing of p53 Gene from a Single Cell1 March 2003 | Clinical Chemistry, Vol. 49, No. 3 Vol. 27, No. 3 Follow us on social media for the latest updates Metrics Downloaded 524 times History Published online 22 August 2018 Published in print September 1999 Information© 2018 Author(s)PDF download

Pore-Size Controlled and Aminated Poly(γ-methyl L-glutamate) Particles for Selective Removal of Nucleic Acids

This report describes a method for selective removal of nucleic acids from various protein solutions, using aminated poly(γ-methyl L-glutamate) (PMLG-NH2) particles. The adsorbing activity for nucleic acids (purified DNA from salmon spermary) increased with increase in either the amino-group content or pore-size (molecular mass exclusion of polysaccharide, Mlim) of the particles. The apparent dissociation constant between the DNA and the particles (amino-group content: 3.5 meq g−1) decreased from 8.2×10−13 mol l−1 (M) to 2.0×10−13 M with increase in pore size from 2×103 to 2×105, at pH 7.0 and an ionic strength of μ=0.05. On the other hand, the adsorbing activity of bovine serum albumin increased with increasing Mlim of the particles, but decreased with increasing ionic strength of the buffer. As a result, when Mlim was 2×103 and amino-group content was 3.5 meq g−1, PMLG-NH2 particles removed DNA from various DNA-containing samples, such as protein solutions, at pH 7.0 and ionic strength (μ) of 0.2. The particles also removed DNA from crude antigen solutions originating from Bordetella bronchiseptica and Pasteurella multocida. A high recovery of protective antigen (100 %) was obtained with each sample solution after removal of the DNA, and the concentration of DNA in it decreased to below 10 ng ml−1.

Nucleic acid and protein elimination during the sugar manufacturing process of conventional and transgenic sugar beets

The fate of cellular DNA during the standard purification steps of the sugar manufacturing process from conventional and transgenic sugar beets was determined. Indigenous nucleases of sugar beet cells were found to be active during the first extraction step (raw juice production) which was carried out at 70°C. This and the consecutive steps of the manufacturing process were validated in terms of DNA degradation by competitive PCR of added external DNA. Each step of the process proved to be very efficient in the removal of nucleic acids. Taken together, the purification steps have the potential to reduce the amount of DNA by a factor of >10 14, exceeding by far the total amount of DNA present in sugar beets. Furthermore, the gene products of the transgenes neomycin phosphotransferase and BNYVV (rhizomania virus) coat protein CP21 were shown to be removed during the purification steps, so that they could not be detected in the resulting white sugar. Thus, sugar obtained from conventional and transgenic beets is indistinguishable or substantially equivalent with respect to purity.

Integrated removal of nucleic acids and recovery of LDH from homogenate of beef heart by affinity precipitation.

Lactate dehydrogenase (LDH) was purified from beef heart homogenate by affinity precipitation. The protein purification was integrated with nucleic acid removal and was done by precipitation of nucleic acids by addition of poly(ethylene imine) PEI onto which a ligand, Cibacron blue, had been coupled. The yield of LDH after elution from the precipitate was 63%, the purification factor 6.9 and the nucleic acid content was reduced by 98%. The capacity of the affinity polymer Cibacron blue-PEI is dependent on the nucleic acid concentration in the homogenate. The beef heart homogenate had an unfavourable ratio of nucleic acids to LDH. Precipitation with recirculated Cibacron blue-PEI, already complexed with some nucleic acids, improved the yield of the enzyme to 74%. The loss of Cibacron blue-PEI, when recirculated, was less than 1% after each cycle.

NH4OH-Based pretreatment for improving the nutritional quality of single-cell protein (SCP)

The potentiality of treatments with NH4OH solutions for improving the quality of protein concentrates fromCandida utilis biomass was studied. The effects of NH4OH concentration, reaction time, and temperature on both biomass recovery and composition of processed samples (including nucleic acid and protein contents) were studied. The results obtained were used to develop empirical models providing a quantitative interpretation of the interrelationships among the variables involved. Additional discussion of the reaction selectivity is provided. Under selected conditions, 96% of nucleic acid removal was achieved with 88% protein recovery. The treated cells were high in vitro digestibility and showed an amino acid profile similar to that of untreated biomass.

Immobilized Nucleases

Nucleases are important analytical enzymes and are used widely for the determination of nucleic acid structure. Their application depends on the specificity and mode of action of the particular enzyme. Nucleases have also found application in the production of flavor enhancers like 5' IMP and 5' GMP, removal of nucleic acids in single cell protein preparations, and as therapeutic agents. Immobilization of nucleases and the use of immobilized nucleases for various biotechnological applications have been reviewed.

5157113 Removal of nucleic acids from monoclonal antibody preparations: George Dove, Gautam Mitra assigned to Miles Inc

5157113 Removal of nucleic acids from monoclonal antibody preparations: George Dove, Gautam Mitra assigned to Miles Inc

5157113 Removal of nucleic acids from monoclonal antibody preparations: George Dove, Gautam Mitra assigned to Miles Inc

5157113 Removal of nucleic acids from monoclonal antibody preparations: George Dove, Gautam Mitra assigned to Miles Inc