Showing papers on "Lysis published in 2013"
TL;DR: It is demonstrated that the addition of histone-DNA complexes to fibrin results in thicker fibers accompanied by improved stability and rigidity, and Therapeutic strategies could be optimized to enhance fibrinolysis in clots containing DNA and histones.
212 citations
TL;DR: This review is focused on the lysis mechanisms employed by tailed double-stranded DNA bacteriophages, where new developments have recently emerged and the membrane-depolarizing holin function still seems to be essential to activate secreted endolysins.
Abstract: Bacteriophages have developed multiple host cell lysis strategies to promote release of descendant virions from infected bacteria. This review is focused on the lysis mechanisms employed by tailed double-stranded DNA bacteriophages, where new developments have recently emerged. These phages seem to use a least common denominator to induce lysis, the so-called holin-endolysin dyad. Endolysins are cell wall-degrading enzymes whereas holins form ‘holes’ in the cytoplasmic membrane at a precise scheduled time. The latter function was long viewed as essential to provide a pathway for endolysin escape to the cell wall. However, recent studies have shown that phages can also exploit the host cell secretion machinery to deliver endolysins to their target and subvert the bacterial autolytic arsenal to effectively accomplish lysis. In these systems the membrane-depolarizing holin function still seems to be essential to activate secreted endolysins. New lysis players have also been uncovered that promote degradation of particular bacterial cell envelopes, such as that of mycobacteria.
184 citations
TL;DR: Chlorine showed the strongest ability to impair the cell integrity with a majority (≥ 88%) of the cells compromised within the first minute and with the cell lysis rates ranging of 0.640-3.82 h(-1) during 1-60 min.
178 citations
TL;DR: In infections of Gram-negative bacteria, lysis is a three step process, with a choice of two effectors for each step, and a third class of lysis protein, the spanin, is required for disruption of the outer membrane.
150 citations
TL;DR: A novel successive reaction kinetics model is developed using the kinetics of the chlorine reaction with cyanobacterial cells and cell-bound toxins and it is shown thatoxin oxidation rates were similar or faster than cell lysis rates in ultrapure water.
97 citations
TL;DR: These findings provide a strategy to improve the lytic activity of phage endolysins based on facilitating their pass through the negatively charged bacterial envelope, and thereby their interaction with the cell wall target, by modulating the net charge of thecell wall-binding modules.
Abstract: Phage endolysins are murein hydrolases that break the bacterial cell wall to provoke lysis and release of phage progeny. Recently, these enzymes have also been recognized as powerful and specific antibacterial agents when added exogenously. In the pneumococcal system, most cell wall associated murein hydrolases reported so far depend on choline for activity, and Cpl-7 lysozyme constitutes a remarkable exception. Here, we report the improvement of the killing activity of the Cpl-7 endolysin by inversion of the sign of the charge of the cell wall-binding module (from -14.93 to +3.0 at neutral pH). The engineered variant, Cpl-7S, has 15 amino acid substitutions and an improved lytic activity against Streptococcus pneumoniae (including multiresistant strains), Streptococcus pyogenes, and other pathogens. Moreover, we have demonstrated that a single 25-μg dose of Cpl-7S significantly increased the survival rate of zebrafish embryos infected with S. pneumoniae or S. pyogenes, confirming the killing effect of Cpl-7S in vivo. Interestingly, Cpl-7S, in combination with 0.01% carvacrol (an essential oil), was also found to efficiently kill Gram-negative bacteria such as Escherichia coli and Pseudomonas putida, an effect not described previously. Our findings provide a strategy to improve the lytic activity of phage endolysins based on facilitating their pass through the negatively charged bacterial envelope, and thereby their interaction with the cell wall target, by modulating the net charge of the cell wall-binding modules.
81 citations
TL;DR: Direct evidence of Ag-NPs penetration into cells of Gram-negative bacterium S. typhimurium and S. aureus showed how different could be responses induced by the same NPs in relatively simple prokaryotic cells and are exert an active influence on their metabolism.
Abstract: Silver nanoparticles possess antibacterial effect for various bacteria; however mechanisms of the interaction between Ag-NPs and bacterial cells remain unclear. The aim of our study was to obtain direct evidence of Ag-NPs penetration into cells of Gram-negative bacterium S. typhimurium and Gram-positive bacterium S. aureus, and to study cell responses to Ag-NPs. The Ag-NPs (most 8–10 nm) were obtained by gas-jet method. S. typhimurium (7.81 × 107 CFU), or S. aureus (8.96 × 107 CFU) were treated by Ag-NPs (0.05 mg/l of silver) in orbital shaker at 190 rpm, 37 °C. Bacteria were sampled at 0.5, 1, 1.5, 2, 5 and 23 h of the incubation for transmission electron microscopy of ultrathin sections. The Ag-NPs adsorbed on outer membrane of S. typhimurium and cell wall of S. auereus; penetrated and accumulated in cells without aggregation and damaging of neighboring cytoplasm. In cells of S. aureus Ag-NPs bound with DNA fibers. Cell responses to Ag-NPs differed morphologically in S. typhimurium and S. aureus, and mainly were presented by damage of cell structures. The cytoplasm of S. aureus became amorphous, while S. typhimurium showed lumping and lysis of cytoplasm which led to formation of “empty” cells. Other difference was fast change of cell shape in S. typhimurium, and late deformation of S. aureus cells. The obtained results showed how different could be responses induced by the same NPs in relatively simple prokaryotic cells. Evidently, Ag-NPs directly interact with macromolecular structures of living cells and are exert an active influence on their metabolism.
65 citations
TL;DR: This study optimised the method for HA extraction, which impacts the assessment of the quantity and quality of PHA biopolymers.
62 citations
TL;DR: An extremely simple and effective colony PCR procedure is established for both gram-negative and gram-positive bacteria, yeasts, and microalgae and Y-PER is observed to be more effective than Tris/EDTA, 0.2 % SDS, and 10 mM EDTA in the extraction of PCR-quality genomic DNA from those microorganisms.
Abstract: An extremely simple and effective colony PCR procedure is established for both gram-negative and gram-positive bacteria, yeasts, and microalgae. Among the four lysis buffers examined, Y-PER is observed to be more effective than Tris/EDTA, 0.2 % SDS, and 10 mM EDTA in the extraction of PCR-quality genomic DNA from those microorganisms. Vortexing or pipetting agitation of the cells in Y-PER for 5–10 s was sufficient to release genomic DNA for all the test bacteria and yeasts, and most microalgae. Additional incubation at 98 °C for 5 min for further cell disruption was essential only for Chlorella vulgaris due to its notoriously rigid cell wall.
56 citations
TL;DR: Bovine serum albumin (BSA) is found to be the best lysis agent, resulting in efficient cell lysis, high RNA stability, and enhanced reverse transcription efficiency, when analyzing from 1 up to 512 mammalian cells.
Abstract: The interest to analyze single and few cell samples is rapidly increasing. Numerous extraction protocols to purify nucleic acids are available, but most of them compromise severely on yield to remove contaminants and are therefore not suitable for the analysis of samples containing small numbers of transcripts only. Here, we evaluate 17 direct cell lysis protocols for transcript yield and compatibility with downstream reverse transcription quantitative real-time PCR. Four endogenously expressed genes are assayed together with RNA and DNA spikes in the samples. We found bovine serum albumin (BSA) to be the best lysis agent, resulting in efficient cell lysis, high RNA stability, and enhanced reverse transcription efficiency. Furthermore, we found direct cell lysis with BSA superior to standard column based extraction methods, when analyzing from 1 up to 512 mammalian cells. In conclusion, direct cell lysis protocols based on BSA can be applied with most cell collection methods and are compatible with most analytical workflows to analyze single-cells as well as samples composed of small numbers of cells.
51 citations
TL;DR: Modifications to the co-extraction protocol improved nucleic acid extraction efficiency from all adsorptive soils and were successfully validated by DGGE analysis of the indigenous community based on 16S rRNA gene and transcripts in soils representing low biomass and/or high clay content.
Abstract: Quantification of microbial gene expression is increasingly being used to study key functions in soil microbial communities, yet major limitations still exist for efficient extraction of nucleic acids, especially RNA for transcript analysis, from this complex matrix. We present an improved extraction protocol that was optimized by: i) including an adsorption-site competitor prior to cell lysis to decrease adsorption of nucleic acids to soil particles, and ii) optimizing the PEG concentration used for nucleic acid precipitation. The extraction efficiency was determined using quantitative real-time PCR on both the RNA (after conversion to cDNA) and the DNA fraction of the extracts. Non-adsorptive soils were characterized by low clay content and/or high phosphate content, whereas adsorptive soils had clay contents above 20% and/or a strong presence of divalent Ca 2+ in combination with high pH. Modifications to the co-extraction protocol improved nucleic acid extraction efficiency from all adsorptive soils and were successfully validated by DGGE analysis of the indigenous community based on 16S rRNA gene and transcripts in soils representing low biomass and/or high clay content. This new approach reveals a robust co-extraction protocol for a range of molecular analysis of diverse soil environments.
TL;DR: In this article, the authors used QIAzolysis reagent (Qiagen) to extract RNA from saliva (both cell-free supernatants and cell pellets), followed by isopropyl alcohol precipitation, cDNA synthesis, and real-time PCR analyses for the genes encoding β-actin (housekeeping) and histatin (a salivary gland-specific gene).
Abstract: BACKGROUND: The use of salivary diagnostics is increasing because of its noninvasiveness, ease of sampling, and the relatively low risk of contracting infectious organisms. Saliva has been used as a biological fluid to identify and validate RNA targets in head and neck cancer patients. The goal of this study was to develop a robust, easy, and cost-effective method for isolating high yields of total RNA from saliva for downstream expression studies. METHODS: Oral whole saliva (200 μL) was collected from healthy controls (n = 6) and from patients with head and neck cancer (n = 8). The method developed in-house used QIAzol lysis reagent (Qiagen) to extract RNA from saliva (both cell-free supernatants and cell pellets), followed by isopropyl alcohol precipitation, cDNA synthesis, and real-time PCR analyses for the genes encoding β-actin (“housekeeping” gene) and histatin (a salivary gland–specific gene). RESULTS: The in-house QIAzol lysis reagent produced a high yield of total RNA (0.89–7.1 μg) from saliva (cell-free saliva and cell pellet) after DNase treatment. The ratio of the absorbance measured at 260 nm to that at 280 nm ranged from 1.6 to 1.9. The commercial kit produced a 10-fold lower RNA yield. Using our method with the QIAzol lysis reagent, we were also able to isolate RNA from archived saliva samples that had been stored without RNase inhibitors at −80 °C for >2 years. CONCLUSIONS: Our in-house QIAzol method is robust, is simple, provides RNA at high yields, and can be implemented to allow saliva transcriptomic studies to be translated into a clinical setting.
TL;DR: The methylated soybean protein and methylated chickpea protein with isoelectric points around pI 8 were prepared by esterifying 83 % of their free carboxyl groups and tested for their interactions with Listeria monocytogenes and Salmonella Enteritidis and exhibited a concentration-dependent inhibitory action against the two studied bacteria.
Abstract: The methylated soybean protein and methylated chickpea protein (MSP and MCP) with isoelectric points around pI 8 were prepared by esterifying 83 % of their free carboxyl groups and tested for their interactions with Listeria monocytogenes and Salmonella Enteritidis. The two substances exhibited a concentration-dependent inhibitory action against the two studied bacteria with a minimum inhibitory concentration of about 100 μg/mL. The IC50 % of the two proteins against L. monocytogenes (17 μg/mL) was comparable to penicillin but comparatively much lower (15 μg/mL) than that of penicillin (85 μg/mL) against S. Enteritidis. The two proteins could inhibit the growth of L. monocytogenes and S. Enteritidis by about 97 and 91 %, respectively, after 6–12 h of incubation at 37 °C. The constituting subunits of MSP (methylated 11S and methylated 7S) were both responsible for its antimicrobial action. Transmission electron microscopy of the protein-treated bacteria showed various signs of cellular deformation. The cationic proteins can electrostatically and hydrophobically interact with cell wall and cell membrane, producing large pores, pore channels and cell wall and cell membrane disintegration, engendering higher cell permeability leading finally to cell emptiness, lysis and death.
TL;DR: Light lees that spent more than one year in barrels were used for ultrasound-assisted yeast lysis in a model wine and, unlike the classical autolysis, ultrasound led to a high cell disruption, and after 20h of ultrasonic treatment, viable cells were hardly found.
TL;DR: A protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates, and for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences is determined.
Abstract: Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.
TL;DR: In this paper, a novel application of sodium laurate (SL) to the shotgun analysis of membrane proteomes was made, where SL was found not only to lyse the membranes and solubilize membrane proteins as efficiently as SDS, but also to be well compatible with trypsin and chymotrypsin.
Abstract: The hydrophobic nature of most membrane proteins severely complicates their extraction, proteolysis and identification. Although detergents can be used to enhance the solubility of the membrane proteins, it is often difficult for a detergent not only to have a strong ability to extract membrane proteins, but also to be compatible with the subsequent proteolysis and mass spectrometric analysis. In this study, we made evaluation on a novel application of sodium laurate (SL) to the shotgun analysis of membrane proteomes. SL was found not only to lyse the membranes and solubilize membrane proteins as efficiently as SDS, but also to be well compatible with trypsin and chymotrypsin. Furthermore, SL could be efficiently removed by phase transfer method from samples after acidification, thus ensuring not to interfere with the subsequent CapLC-MS/MS analysis of the proteolytic peptides of proteins. When SL was applied to assist the digestion and identification of a standard protein mixture containing bacteriorhodoposin and the proteins in rat liver plasma membrane-enriched fractions, it was found that, compared with other two representative enzyme- and MS-compatible detergents RapiGest SF (RGS) and sodium deoxycholate (SDC), SL exhibited obvious superiority in the identification of membrane proteins particularly those with high hydrophobicity and/or multiple transmembrane domains.
17 Jan 2013
TL;DR: A simple microfluidic thermal lysis device is used to quickly release intracellular nucleic acids and proteins without the need for additional reagents or beads used in traditional chemical or mechanical methods.
Abstract: Development of new diagnostic platforms that incorporate lab-on-a-chip technologies for portable assays is driving the need for rapid, simple, low cost methods to prepare samples for downstream processing or detection. An important component of the sample preparation process is cell lysis. In this work, a simple microfluidic thermal lysis device is used to quickly release intracellular nucleic acids and proteins without the need for additional reagents or beads used in traditional chemical or mechanical methods (e.g., chaotropic salts or bead beating). On-chip lysis is demonstrated in a multi-turn serpentine microchannel with external temperature control via an attached resistive heater. Lysis was confirmed for Escherichia coli by fluorescent viability assay, release of ATP measured with bioluminescent assay, release of DNA measured by fluorometry and qPCR, as well as bacterial culture. Results comparable to standard lysis techniques were achievable at temperatures greater than 65 °C and heating durations between 1 and 60 s.
TL;DR: The results suggested that anammox bacteria contain peptidoglycan-like components in their cell wall that can be targeted by lysozyme and penicillin G-sensitive proteins were involved in their synthesis.
Abstract: Anaerobic ammonium-oxidizing (anammox) planctomycetes oxidize ammonium in the absence of molecular oxygen with nitrite as the electron acceptor. Although planctomycetes are generally assumed to lack peptidoglycan in their cell walls, recent genome data imply that the anammox bacteria have the genes necessary to synthesize peptidoglycan-like cell wall structures. In this study, we investigated the effects of two antibacterial agents that target the integrity and synthesis of peptidoglycan (lysozyme and penicillin G) on the anammox bacterium Kuenenia stuttgartiensis. The effects of these compounds were determined in both short-term batch incubations and long-term (continuous-cultivation) growth experiments in membrane bioreactors. Lysozyme at 1 g/liter (20 mM EDTA) lysed anammox cells in less than 60 min, whereas penicillin G did not have any observable short-term effects on anammox activity. Penicillin G (0.5, 1, and 5 g/liter) reversibly inhibited the growth of anammox bacteria in continuous-culture experiments. Furthermore, transcriptome analyses of the penicillin G-treated reactor and the control reactor revealed that penicillin G treatment resulted in a 10-fold decrease in the ribosome levels of the cells. One of the cell division proteins (Kustd1438) was downregulated 25-fold. Our results suggested that anammox bacteria contain peptidoglycan-like components in their cell wall that can be targeted by lysozyme and penicillin G-sensitive proteins were involved in their synthesis. Finally, we showed that a continuous membrane reactor system with free-living planktonic cells was a very powerful tool to study the physiology of slow-growing microorganisms under physiological conditions.
TL;DR: In the protocol presented here, the recombinant plasmid expressing T7 RNA polymerase (RNAP) as a his6-tagged molecule is under an isopropyl β-d-1-thio-galactopyranoside (IPTG)-inducible promoter that can be stored for months or years at -20°C.
Abstract: For large-scale transcription reactions or for cost savings, a laboratory may want to prepare its own recombinant T7-, SP6-, or T3-phage RNA polymerases. It is convenient to perform this preparation every 2-3 years and have a consistent and reliable source of phage RNA polymerase for many in vitro transcription reactions. In the protocol presented here, the recombinant plasmid expressing T7 RNA polymerase (RNAP) as a his6-tagged molecule is under an isopropyl β-d-1-thio-galactopyranoside (IPTG)-inducible promoter. The bacteria are lysed by sonication, the his6-tagged protein in the bacterial lysate is purified by binding to Ni-NTA agarose, and the resin is then extensively washed and eluted with imidazole. The purified enzyme is dialyzed against a glycerol-containing storage buffer and can then be stored for months or years at -20°C.
TL;DR: Compared with traditional methods of cell disruption, the inducible cell lysis systems are more economically feasible and easier to control and show a promising perspective in industrial production of bio-based chemicals.
Abstract: The release of products from microbial cells is an essential process for industrial scale production of bio-based chemicals. However, traditional methods of cell lysis, e.g., mechanical disruption, chemical solvent extraction, and immobilized enzyme degradation, account for a large share of the total production cost. Thus, an efficient cell lysis system is required to lower the cost. This review has focused on our current knowledge of two cell lysis systems, bacteriophage holin-endolysin system, and lipid enzyme hydrolysis system. These systems are controlled by conditionally inducible regulatory apparatus and applied in microbial production of fatty acids and polyhydroxyalkanoates. Moreover, toxin-antitoxin system is also suggested as alternative for its potential applications in cell lysis. Compared with traditional methods of cell disruption, the inducible cell lysis systems are more economically feasible and easier to control and show a promising perspective in industrial production of bio-based chemicals.
TL;DR: Two different systems that release bacterial compounds have been described and these are dependent of the expression of the colicin lysis genes known for releasing cytoplasmic colicins as well as other soluble proteins.
TL;DR: This unit presents basic procedures for extraction of DNA from both fresh tissues and formalin‐fixed, paraffin‐embedded tissue samples, and two quite different methodologies are described: silica spin column and phenol/chloroform extraction.
Abstract: RNA can be extracted from cultured cells, peripheral blood, bone marrow, plasma, serum, body fluids, and fresh or frozen tissues. RNA can also be extracted from formalin-fixed paraffin-embedded (FFPE) tissues. Methods for RNA extraction can be divided into three groups: phenol/chloroform extraction, silica spin-column absorption, and isopycnic gradient centrifugation. Two different RNA isolation procedures are described in this unit. The first basic protocol describes a one-step isolation involving monophasic lysis with guanidine isothiocyanate and phenol followed by chloroform extraction. The second basic protocol describes a silica-column separation method for RNA isolation.
TL;DR: Salicylic acid-coated magnetic nanoparticles were prepared via a modified one-step synthesis and used for a one-stop extraction of genomic DNA from mammalian cells and the established method is fast, simple, reliable, and environmentally friendly.
TL;DR: It is shown, that the cell lysis and preparation method conserves protein structures well and is suitable for visual analysis by TEM.
TL;DR: Analysis of uptake and individual endosome lysis events in fibroblast, normal, and carcinoma cell lines using a colloidal mesoporous silica (CMS) nanoparticle (NP)-based reporter system with a covalently attached photosensitizer indicates that Renca cells not only take up a lower amount of NPs in comparison with the fibro Blast cells but also have larger endosomes and a lower NP load per endosomal.
Abstract: We investigated uptake and individual endosome lysis events in fibroblast, normal, and carcinoma cell lines using a colloidal mesoporous silica (CMS) nanoparticle (NP)-based reporter system with a covalently attached photosensitizer. Endosome lysis was induced through the activation of protoporphyrin IX (PpIX). Surprisingly, this release-on-demand system resulted in more broadly distributed lysis times than expected, particularly for Renca, a renal carcinoma cell line. An analysis of the NP load per endosome, endosome size, and uptake characteristics indicate that Renca cells not only take up a lower amount of NPs in comparison with the fibroblast cells but also have larger endosomes and a lower NP load per endosome. We then created a stochastic model detailing steps downstream of uptake to understand how much factors that cannot be directly measured, such as variations in the PpIX load per NP, affect the lysis time distributions. Model results indicate that the distributions are primarily determined by t...
TL;DR: A central role for VP4 acting as a viroporin in the disruption of cellular membranes to trigger SV40 viral release by forming toroidal pores that unite the outer and inner leaflets of membrane bilayers is supported.
Abstract: Nonenveloped viruses are generally released from the cell by the timely lysis of host cell membranes. SV40 has been used as a model virus for the study of the lytic nonenveloped virus life cycle. The expression of SV40 VP4 at later times during infection is concomitant with cell lysis. To investigate the role of VP4 in viral release and its mechanism of action, VP4 was expressed and purified from bacteria as a fusion protein for use in membrane disruption assays. Purified VP4 perforated membranes as demonstrated by the release of fluorescent markers encapsulated within large unilamellar vesicles or liposomes. Dynamic light scattering results found that VP4 treatment did not cause membrane lysis or change the size of the liposomes. Liposomes encapsulated with bodipy-labeled streptavidin were used to show that VP4 formed stable pores in membranes. These VP4 pores had an inner diameter of between 1 and 5 nm. Asymmetrical liposomes containing pyrene-labeled lipids in the outer monolayer were employed to monitor transbilayer lipid diffusion. Consistent with VP4 forming toroidal pore structures in membranes, VP4 induced transbilayer lipid diffusion or lipid flip-flop. Altogether, these studies support a central role for VP4 acting as a viroporin in the disruption of cellular membranes to trigger SV40 viral release by forming toroidal pores that unite the outer and inner leaflets of membrane bilayers.
TL;DR: A novel insight is presented into the mechanism of the lysis process of a thermophile bacterium by its phage at high temperatures, which should be helpful in revealing the roles of thermophilic bacteriophages in the biosphere of deep-sea hydrothermal vents.
Abstract: The holin–endolysin system is used by double-stranded DNA phages to lyse their bacterial hosts at the terminal stage of the phage reproduction cycle. Endolysins are proteins with one of several muralytic activities able to digest the bacterial cell wall for phage progeny release. However, the functions of thermophilic bacteriophage endolysin in host lysis have not been extensively investigated. In this study, the roles of the endolysin of a thermophilic bacteriophage, GVE2, from a deep-sea hydrothermal vent, which could infect Geobacillus sp. E263 at high temperatures, were characterized. The results showed that GVE2 could lead to lysis of host cells. The confocal microscopy data showed that GFP–endolysin aggregated in GVE2-infected Geobacillus sp. E263 cells, showing the involvement of endolysin in the lysis process at high temperatures. The results revealed that the GVE2 endolysin and holin interacted directly. It was found that the endolysin could interact with the host protein ABC transporter, suggesting that host proteins might participate in the regulation of the lysis process. Therefore, our study presents a novel insight into the mechanism of the lysis process of a thermophilic bacterium by its phage at high temperatures, which should be helpful in revealing the roles of thermophilic bacteriophages in the biosphere of deep-sea hydrothermal vents.
TL;DR: It is demonstrated that APols added for cell lysis help maintain the proteome in solution, are compatible with protein digestion using trypsin, and can readily be removed prior to mass spectrometry by a one-step acidification and centrifugation.
Abstract: In proteomics, detergents and chaotropes are indispensable for proteome analysis, not only for protein extraction, but also for protein digestion To increase the protein extraction efficiency, detergents are usually added in the lysis buffer to extract membrane proteins out of membrane structure and to maintain protein in solutions In general, these detergents need to be removed prior to protein digestion, usually by precipitation or ultrafiltration Digestion often takes place in the presence of chaotropic reagents, such as urea, which often need to be removed prior to mass spectrometry The addition and removal of detergents and chaotropes require multiple steps that are time-consuming and can cause sample losses Amphipols (APols) are a different class of detergents that have physical and solubilization properties that are distinct from conventional detergents They have primarily been used in protein structure analysis for membrane protein trapping and stabilization Here, we demonstrate a simple and rapid protocol for total and membrane proteome preparation using APols We demonstrate that APols added for cell lysis help maintain the proteome in solution, are compatible with protein digestion using trypsin, and can readily be removed prior to mass spectrometry by a one-step acidification and centrifugation This protocol is much faster, can be performed in a single tube, and can readily replace the conventional detergent/chaotrope approaches for total and membrane proteome analysis
TL;DR: The high yield expression of the human LAT1 transporter has been obtained for the first time using E. coli using the Rosetta(DE3)pLysS strain ofE.
Abstract: The high yield expression of the human LAT1 transporter has been obtained for the first time using E. coli. The hLAT1 cDNA was amplified from HEK293 cells and cloned in pH6EX3 vector. The construct pH6EX3-6His-hLAT1 was used to express the 6His-hLAT1 protein in the Rosetta(DE3)pLysS strain of E. coli. The highest level of expression was detected 8 h after induction by IPTG at 28 °C. The expressed protein was collected in the insoluble fraction of cell lysate. On SDS-PAGE the apparent molecular mass of the polypeptide was 40 kDa. After solubilization with sarkosyl and denaturation with urea the protein carrying a 6His N-terminal tag was purified by Ni(2+)-chelating affinity chromatography and identified by anti-His antibody. The yield of the over-expressed protein after purification was 3.5 mg/L (cell culture). The human CD98 cDNA amplified from Imagene plasmid was cloned in pGEX-4T1. The construct pGEX-4T1-hCD98 was used to express the GST-hCD98 protein in the Rosetta(DE3)pLysS strain of E. coli. The highest level of expression was detected in this case 4 h after induction by IPTG at 28 °C. The expressed protein was accumulated in the soluble fraction of cell lysate. The molecular mass was determined on the basis of marker proteins on SDS-PAGE; it was about 110 kDa. GST was cleaved from the protein construct by incubation with thrombin for 12 h and the hCD98 was separated by Sephadex G-200 chromatography (size exclusion). hCD98 showed a 62 kDa apparent molecular mass, as determined on the basis of molecular mass markers using SDS-PAGE. The yield of CD98 was 2 mg/L of cell culture.
TL;DR: The hypothesis that the use of nanoparticles with increased densities can provide enhanced enrichment of plasma membrane proteins for proteomic analysis is tested.
Abstract: Proteomic and other characterization of plasma membrane proteins is made difficult by their low abundance, hydrophobicity, frequent carboxylation, and dynamic population. We and others have proposed that underrepresentation in LC-MS/MS analysis can be partially compensated by enriching the plasma membrane and its proteins using cationic nanoparticle pellicles. The nanoparticles increase the density of plasma membrane sheets and thus enhance separation by centrifugation from other lysed cellular components. Herein, we test the hypothesis that the use of nanoparticles with increased densities can provide enhanced enrichment of plasma membrane proteins for proteomic analysis. Multiple myeloma cells were grown and coated in suspension with three different pellicles of three different densities and both pellicle coated and uncoated suspensions analyzed by high-throughput LC-MS/MS. Enrichment was evaluated by the total number and the spectral counts of identified plasma membrane proteins.