Purification of his-tagged proteins by immobilized chelate affinity chromatography: the benefits from the use of organic solvent

Recombinant proteins overexpressed in and purified from Escherichia coli contain impurities that are toxic in biological assays. The application of affinity purification procedures is often not sufficient to remove these toxic components. We here describe a simple and fast, one-step protocol to remove these impurities highly efficiently. Four recombinant proteins were overexpressed in E. coli as His-tagged fusion proteins and purified by immobilized metal chelate affinity chromatography on Ni-NTA beads. Depending on the protein, the composition of the lysis buffer, and the washing protocol, various impurities appeared to be present in the purified protein preparations. Here we show how the use of 60% isopropanol during washing steps removed most of these contaminants from the end products. In addition to the removal of proteins that aspecifically adhere to the beads or to the tagged protein, this procedure was particularly useful in removing endotoxins. Moreover, we show that detergents such as NP-40, that are necessarily employed during lysis, are also efficiently removed. Finally, we show that proteins are able to refold correctly after isopropanol treatment. Thus, the resulting end products contain significantly less contaminating E. coli proteins, endotoxins, and detergents.     

Measurement of prompt DNA double-strand breaks in mammalian cells without including heat-labile sites: results for cells deficient in nonhomologous end joining

Ionizing radiation induces prompt single-strand breaks and double-strand breaks in DNA. In addition, labile sites are induced that can be converted to breaks by heat or mild alkali. When such labile lesions are present within multiply damaged sites, additional double-strand breaks can form. Current protocols for measurement of DNA double-strand breaks involve a lysis step at an elevated temperature, and consequently breaks from heat-labile sites will be generated during lysis and will be included in the measurement. However, such sites may not develop into breaks within the cell and therefore may not need DNA double-strand break repair processes for elimination. We present here a new lysis and pulsed-field gel electrophoresis protocol that is carried out entirely at 0-4 degrees C and thus avoids inclusion of heat-labile sites in the measurement. The new recommended lysis procedure involves two steps: The first step includes proteinase K, which has sufficient activity at 0 degrees C to support lysis, and the second step includes a high-salt buffer to further free the DNA from proteins and other cellular structures. Using various tests, we conclude that lysis is sufficient with this procedure to allow accurate determination of double-strand breaks by pulsed-field gel electrophoresis. Using the new protocol, it was found that heat-labile sites account for 30% of the initial number of double-strand breaks measured by conventional protocols after exposure to low-LET radiation. In addition, we show that heat-labile sites that can be converted to double-strand breaks are repaired with fast kinetics and are almost completely eliminated after 1 h at 37 degrees C. A study of cells deficient in nonhomologous end joining reveals that the residual fast repair response typically seen in such cells is solely due to repair at heat-labile sites and is not due to repair of prompt DSBs.     

Colony filtration blotting for screening soluble expression in Escherichia coli

We have developed a screen for detecting E. coli colonies that produce soluble recombinant target proteins at the colony level: the colony filtration (CoFi) blot. Colonies are transferred, induced and lysed on a filter membrane that can separate soluble proteins from inclusion bodies. Upon lysis, the soluble proteins diffuse through the filter membrane and are captured on a nitrocellulose membrane. The nitrocellulose membrane is incubated with antibodies or probes specific for the target protein and are then developed. In the resulting image, colonies expressing soluble protein can easily be identified. This protocol can be used to screen thousands of constructs in a matter of days, making it very suitable for expression libraries. The protocol is robust and flexible with regard to lysis conditions, induction temperatures and strains. The method requires only standard laboratory equipment and is based on immunochemicals used for western blotting. The following protocol describes the screening of a DNA library with detection done using chemiluminescence. Depending on induction temperature, the whole procedure can be performed in <2 d.     

The function of lymphocyte proteases. Inhibition and restoration of granule-mediated lysis with isocoumarin serine protease inhibitors.

To kill other cells, lymphocytes can exocytose granules that contain serine proteases and pore-forming proteins (perforins). We report that mechanism-based isocoumarin inhibitors inhibited the proteases and inactivated lysis. When inhibited proteases were restored, lysis was also restored, indicating that the proteases were essential for lysis. We found three new lymphocyte protease activities, "Asp-ase,"Met-ase," and "Ser-ase," which in addition to ly-tryptase and ly-chymase, comprise five different protease activities in rat RNK-16 granules. The general serine protease inhibitor 3,4-dichloroisocoumarin (DCI) inhibited all five protease activities. Essentially all protease molecules were inactivated by DCI before lysis was reduced, as determined from DCI's second order inhibition rate constants for the proteases, the DCI concentrations, and the times of pretreatment needed to block lysis. The pH favoring DCI inhibition of lysis was the pH optimum for protease activity. Isocoumarin reagents acylate, and may sometimes secondarily alkylate, serine protease active sites. Granule proteases, inhibited by DCI acylation, were deacylated with hydroxylamine, restoring both the protease and lytic activities. Hydroxylamine does not restore alkylated proteases and did not restore the lytic activities after inhibition with 4-chloro-7-guanidino-3-(2-phenylethoxy)-isocoumarin, a more alkylating mechanism-based inhibitor designed to react with tryptases. It is improbable that isocoumarin reagents directly inactivated pore-forming proteins because 1) these reagents require protease activation, 2) their nonspecific effects are alkylating, and 3) alkylated proteins are not restored by hydroxylamine. We conclude that serine proteases participate in lysis when lysis is mediated by the complete assembly of granule proteins.     

Localization of DnaK (chaperone 70) from Escherichia coli in an osmotic-shock-sensitive compartment of the cytoplasm.

The chaperone DnaK can be released (up to 40%) by osmotic shock, a procedure which is known to release the periplasmic proteins and a select group of cytoplasmic proteins (including thioredoxin and elongation factor Tu) possibly associated with the inner face of the inner membrane. As distinct from periplasmic proteins, DnaK is retained within spheroplasts prepared with lysozyme and EDTA. The ability to isolate DnaK with a membrane fraction prepared under gentle lysis conditions supports a peripheral association between DnaK and the cytoplasmic membrane. Furthermore, heat shock transiently increases the localization of DnaK in the osmotic-shock-sensitive compartment of the cytoplasm. We conclude that DnaK belongs to the select group of cytoplasmic proteins released by osmotic shock, which are possibly located at Bayer adhesion sites, where the inner and outer membranes are contiguous.     

A rapid chemical procedure for isolation and purification of chromosomal DNA from gram-negative bacilli

A rapid and simple method for preparing chromosomal DNA from gram-negative bacilli is presented. It is based on the alkaline (NaOH 0.03 m) lysis of cell walls. The resulting emulsion is purified by proteinase K (0.625 mg/g of wet wt), SDS, and the deproteinizing agent (chloroform isoamyl alcohol). The purity, molecular nature, and yield of DNA obtained by the present method are compared with those of DNA extracted by Marmur's procedure and a Marmur's modified procedure. We have developed and standardized this original method to isolate double-stranded DNA, free of proteins and RNA contamination and with a significantly higher yield of DNA than the two other methods. This procedure is particularly useful for strains with low growth and can be applied in every field concerned with DNA analysis.     

In-cell NMR spectroscopy

Structural studies by in-cell nuclear magnetic resonance are a developing new field of research, and their objective is to obtain structural information of proteins and other biological macromolecules in the cytoplasm of Escherichia coli cells. The major limitation of in-cell experiments is cell lysis that occurs during the experiments. In this article, we describe how inhibition of autologous expression by rifampicin at a high concentration decreases cell lysis in E. coli. We suggest that rifampicin is acting in the programmed cell death gene system MazEF, which is triggered by stress conditions and ultimately leads to cell lysis.     

Insights into a Viral Lytic Pathway from an Archaeal Virus-Host System

Archaeal host cells infected by Sulfolobus turreted icosahedral virus (STIV) and Sulfolobus islandicus rod-shaped virus 2 (SIRV2) produce unusual pyramid-like structures on the cell surface prior to virus-induced cell lysis. This viral lysis process is distinct from known viral lysis processes associated with bacterial or eukaryal viruses. The STIV protein C92 and the SIRV2 protein 98 are the only viral proteins required for the formation of the pyramid lysis structures of STIV and SIRV2, respectively. Since SIRV2 and STIV have fundamentally different morphotypes and genome sequences, it is surprising that they share this lysis system. In this study, we have constructed a collection of C92/P98 chimeric proteins and tested their abilities, both in the context of virus replication and alone, to form pyramid lysis structures in S. solfataricus. The results of this study illustrate that these proteins are functionally homologous when expressed as individual chimeric proteins but not when expressed in the context of complete STIV infection.     

Potential role of cellular ESCRT proteins in the STIV life cycle

We are examining the archaeal virus STIV (Sulfolobus turreted icosahedral virus) in order to elucidate the details of its replication cycle and its interactions with its cellular host, Sulfolobus solfataricus. Infection of Sulfolobus by STIV initiates an unusual cell lysis pathway. One component of this pathway is the formation of pyramid-like structures on the surface of infected cells. Multiple seven-sided pyramid-like structures are formed on infected cells late in the STIV replication cycle. These pyramid-like structures are formed at sites where the Sulfolobus S-layer has been disrupted and through which the cellular membrane protrudes. It is through the pyramid-like structures that virus-induced cell lysis occurs in the final stages of the STIV replication cycle. The pathway and process by which these unusual lysis structures are produced appears to be novel to archaeal viruses and are not related to the well-characterized lysis mechanisms utilized by bacterial viruses. We are interested in elucidating both the viral and cellular components involved with STIV lysis of its infected cell. In particular, we are examining the potential role that Sulfolobus ESCRT (endosomal sorting complex required for transport)-like proteins play during viral infection and lysis. We hypothesize that STIV takes advantage of the Sulfolobus ESCRT machinery for virus assembly, transport and cellular lysis.     

Rapid sedimentation of Escherichia coli in the presence of polyethylene glycol at 1g

In order to harvest a 100-liter culture of bacteria with a conventional refrigerated centrifuge, using a 1.5- or 3.0-liter capacity rotor, about 10 hr of arduous work are involved. In contrast, the procedure described using PEG results in almost quantitative recovery of viable cells, with the expenditure of less than 10% of the labor. This technique may be applied to any desired volume of culture using simple equipment which may be built in any laboratory. It should be applicable for harvesting E. coli destined for the isolation of any stable protein, nucleic acid, or other component, including bacteriophage which carry a late lysis mutation thus preventing lysis of bacterial cells; however, this procedure may not be suitable for proteins which are not stable at 4°C for 12 hr.     

Investigating the two regimes of fibrin clot lysis: an experimental and computational approach

It has been observed in vitro that complete clot lysis is generally preceded by a slow phase of lysis during which the degradation seems to be inefficient. However, this slow regime was merely noticed, but not yet quantitatively discussed. In our experiments, we observed that the lysis ubiquitously occurred in two distinct regimes, a slow and a fast lysis regime. We quantified extensively the duration of these regimes for a wide spectrum of experimental conditions and found that on average, the slow regime lasts longer than the fast one, meaning that during most of the process, the lysis is ineffective. We proposed a computational model in which the properties of the binding of the proteins change during the lysis: first, the biochemical reactions take place at the surface of the fibrin fibers, then in the bulk, resulting in the observed fast lysis regime. This simple hypothesis appeared to be sufficient to reproduce with a great accuracy the lysis profiles obtained experimentally.     

Cloned cytotoxic T lymphocytes as target cells. II. Polarity of lysis revisited

The original polarity of lysis experiments suggested that CTL are themselves sensitive to whatever mechanism it is that CTL use to lyse their targets. This concept has placed certain limitations on possible mechanisms of lysis by CTL. Recently, we found in studies with cloned CTL as targets that cloned CTL are in fact highly resistant to lysis by other CTL, as well as to their cytotoxic granule proteins. We show here that although cloned CTL are extremely resistant to lysis by primary and cloned CTL, they are readily inactivated functionally by all primary CTL and by at least one CTL clone. Moreover, cloned CTL are also functionally inactivated by cytotoxic granule proteins. The activation of CTL, which we call inhibitin, is Ca2+ insensitive and distinct from hemolytic activity, and is, thus, unlikely to be perforin. These experiments suggest a possible alternative interpretation of the original polarity of lysis experiments.     

Study of lipid rafts by gel filtration combined with preliminary staining with fluorescently labeled antibodies

A novel procedure for detecting raft-associated proteins by gel filtration was developed. Cells were stained with fluorescently labeled protein-specific antibodies, lyzed in a nonionic detergent and gel-filtered on Sepharose 4B. Proteins were identified with the help of fluorescently labeled antibodies. Staining of cells with fluorescently labeled antibodies prior to cell lysis significantly simplified the identification procedure. The potentialities of the new approach are demonstrated in the example of several surface proteins associated with lipid rafts either constitutively or in the course of cell activation.     

Clocking out: modeling phage-induced lysis of Escherichia coli

Phage lambda lyses the host Escherichia coli at a precisely scheduled time after induction. Lysis timing is determined by the action of phage holins, which are small proteins that induce hole formation in the bacterium's cytoplasmic membrane. We present a two-stage nucleation model of lysis timing, with the nucleation of condensed holin rafts on the inner membrane followed by the nucleation of a hole within those rafts. The nucleation of holin rafts accounts for most of the delay of lysis after induction. Our simulations of this model recover the accurate lysis timing seen experimentally and show that the timing accuracy is optimal. An enhanced holin-holin interaction is needed in our model to recover experimental lysis delays after the application of membrane poison, and such early triggering of lysis is possible only after the inner membrane is supersaturated with holin. Antiholin reduces the delay between membrane depolarization and lysis and leads to an earlier time after which triggered lysis is possible.     

Another turn of the screw in shaving Gram-positive bacteria: Optimization of proteomics surface protein identification in Streptococcus pneumoniae

Bacterial surface proteins are of outmost importance as they play critical roles in the interaction between cells and their environment. In addition, they can be targets of either vaccines or antibodies. Proteomic analysis through "shaving" live cells with proteases has become a successful approach for a fast and reliable identification of surface proteins. However, this protocol has not been able to reach the goal of excluding cytoplasmic contamination, as cell lysis is an inherent process during culture and experimental manipulation. In this work, we carried out the optimization of the "shaving" strategy for the Gram-positive human pathogen Streptococcus pneumoniae, a bacterium highly susceptible to autolysis, and set up the conditions for maximizing the identification of surface proteins containing sorting or exporting signals, and for minimizing cytoplasmic contamination. We also demonstrate that cell lysis is an inherent process during culture and experimental manipulation, and that a low level of lysis is enough to contaminate a "surfome" preparation with peptides derived from cytoplasmic proteins. When the optimized conditions were applied to several clinical isolates, we found the majority of the proteins described to induce protection against pneumococcal infection. In addition, we found other proteins whose protection capacity has not been yet tested. In addition, we show the utility of this approach for providing antigens that can be used in serological tests for the diagnosis of pneumococcal disease.     

An automated small-scale protein expression and purification screening provides beneficial information for protein production

One of the first key steps in structural genomics is high-throughput expression and rapid screening to select highly soluble proteins, the preferred candidates for crystal production. Here we describe the methodology used at the Berkeley Structural Genomics Center (BSGC) for automated parallel expression and small-scale purification of fusion proteins using a 96-well format. Our robotic method includes cell lysis, soluble fraction separation and purification with affinity resins. For detection of His-tagged proteins in the soluble fractions and after affinity resin elution, a dot-blot procedure with an anti-His-antibody is used. The expression level and molecular mass of recombinant proteins are checked by SDS-PAGE. With this approach, we are able to obtain beneficial information to be used for large-scale protein expression and purification.     

Staphylococcus aureus CidA and LrgA proteins exhibit holin-like properties

The Staphylococcus aureus cid and lrg operons are known to be involved in biofilm formation by controlling cell lysis and the release of genomic DNA, which ultimately becomes a structural component of the biofilm matrix. Although the molecular mechanisms controlling cell death and lysis are unknown, it has been hypothesized that the cidA and lrgA genes encode holin- and antiholin-like proteins and function to regulate these processes similarly to bacteriophage-induced death and lysis. In this study, we focused on the biochemical and molecular characterization of CidA and LrgA with the goal of testing the holin model. First, membrane fractionation and fluorescent protein fusion studies revealed that CidA and LrgA are membrane-associated proteins. Furthermore, similarly to holins, CidA and LrgA were found to oligomerize into high-molecular-mass complexes whose formation was dependent on disulfide bonds formed between cysteine residues. To determine the function of disulfide bond-dependent oligomerization of CidA, an S. aureus mutant in which the wild-type copy of the cidA gene was replaced with the cysteine mutant allele was generated. As determined by β-galactosidase release assays, this mutant exhibited increased cell lysis during stationary phase, suggesting that oligomerization has a negative impact on this process. When analyzed for biofilm development and maturation, this mutant displayed increased biofilm adhesion in a static assay and a greater amount of dead-cell accumulation during biofilm maturation. These studies support the model that CidA and LrgA proteins are bacterial holin-/antiholin-like proteins that function to control cell death and lysis during biofilm development.     

Mesenchymal Stem Cells Engineered to Inhibit Complement-Mediated Damage

Mesenchymal stem cells (MSC) preferentially migrate to damaged tissues and, due to their immunomodulatory and trophic properties, contribute to tissue repair. Although MSC express molecules, such as membrane cofactor protein (CD46), complement decay-accelerating factor (CD55), and protectin (CD59), which confer protection from complement-mediated lysis, MSC are recruited and activated by anaphylatoxins after transplantation, potentially causing MSC death and limiting therapeutic benefit. We have previously demonstrated that transduction of MSC with a retrovirus encoding HCMV-US proteins resulted in higher levels of MSC engraftment due to decreased HLA-I expression. Here, we investigate whether engineering MSC to express US2 (MSC-US2), US3 (MSC-US3), US6 (MSC-US6), or US11 (MSC-US11) HCMV proteins can alter complement recognition, thereby better protecting MSC from complement attack and lysis. HCMV-US proteins increased MSC CD59 expression at different levels as determined by flow cytometric evaluation of the median fluorescence intensity ratio (MFI). A significant increase in CD59 expression was seen in MSC-US2, MSC-US3, and MSC-US6, but not in MSC-US11. Only MSC-US2 displayed increased expression of CD46, while US2 and US3 proteins were both able to augment the percentage of MSC expressing this molecule. Regardless of the HCMV protein expressed, none changed CD55 MFI; however, expression of US6, US11, and US2 each increased the percentage of MSC that were positive for this molecule. Because US2 protein was the most efficient in up-regulating all three complement regulatory proteins, we used a functional complement-mediated cytotoxicity assay to investigate whether MSC-US2 were protected from complement-mediated lysis. We demonstrated that over-expression of the US2 protein reduced complement lysis by 59.10±12.89% when compared to untransduced MSC. This is the first report, to our knowledge, describing a role of HCMV-US proteins in complement evasion, and our data shows that over-expression of US2 protein on MSC could serve as a strategy to protect these cells from complement lysis.     

HPLC-MS/MS shotgun proteomic research of deer antlers with multiparallel protein extraction methods

Deer antlers mature rapidly in 60 days, and subsequently shed in 5 days with rapid ossification. During this procedure, the function of deer antlers changes significantly. Therefore, the profiling of antler proteome is helpful to discover important growing and shedding regulation proteins, which might be of great significance for studying development and regeneration. In this study, a parallel protein extraction strategy was developed to extract proteins from antlers of red deer with five different lysis solutions, followed by shotgun proteomic analysis by microflow reversed-phase liquid chromatography/electrospray ionization/tandem mass spectrometry (μRPLC-ESI-MS/MS) with a 30 cm-long serially coupled microcolumn. Our experimental results showed that the identified proteins extracted by five kinds of lysis solution were complementary to each other. In total, 416 unique proteins were identified, with relative molecular masses from 2000 to 600,000, and isoelectric points from 3.84 to 11.57. All these results demonstrate that the combination of parallel protein extraction strategy and μRPLC-ESI-MS/MS analysis with serially coupled long microcolumns might be of great significance for comprehensive proteomic research of deer antler.