Clarified Cell Lysates Research Articles

ViriChip: a solid phase assay for detection and identification of viruses by atomic forcemicroscopy

Bionanotechnology can be viewed as the integration of tools and concepts innanotechnology with the attributes of biomolecules. We report here on an atomic forcemicroscopy–immunosensor assay (AFMIA) that couples AFM with solid phase affinitycapture of biological entities for the rapid detection and identification of group Bcoxsackievirus particles. Virus identification is based on type-specific immunocapture andthe morphological properties of the captured viruses as obtained by the AFM.Representatives of the six group B coxsackieviruses have been specifically captured from1 µl volumes of clarified cell lysates, body fluids and environmental samples.Concentration and kinetic profiles for capture indicate that detection is possible at103 TCID50 µl−1 and the dynamic range of the assay spans three logs. The results demonstrate thatthe melding of a nanotechnological tool (AFM) with biotechnology (solid phaseimmunocapture of virus particles) can create a clinically relevant platform, useful forthe detection and identification of enterovirus particles in a variety of samples.

Protein-mediated isolation of plasmid DNA by a zinc finger-glutathione S-transferase affinity linker.

The sequence-specific affinity chromatographic isolation of plasmid DNA from crude lysates of E. coli DH5alpha fermentations is addressed. A zinc finger-GST fusion protein that binds a synthetic oligonucleotide cassette containing the appropriate DNA recognition sequence is described. This cassette was inserted into the SmaI site of pUC19 to enable the affinity isolation of the plasmid. It is shown that zinc finger-GST fusion proteins can bind both their DNA recognition sequence and a glutathione-derivatized solid support simultaneously. Furthermore, a simple procedure for the isolation of such plasmids from clarified cell lysates is demonstrated. Cell lysates were clarified by cross-flow Dean vortex microfiltration, and the permeate was incubated with zinc finger-GST fusion protein. The resulting complex was adsorbed directly onto glutathione-Sepharose. Analysis of the glutathione-eluted complex showed that plasmid DNA had been recovered, largely free from contamination by genomic DNA or bacterial cell proteins.

Recovering antibody secretion using a hapten ligand as a chemical chaperone.

Engineered antibodies have come to the forefront as research reagents and clinical therapeutics. However, reduced stability or expression levels pose a major problem with many engineered antibodies. As a model for understanding functional consequences of variable region mutation, we have studied the assembly and trafficking of anti-phenylphosphocholine antibodies. Previously, we identified severe secretion defects because of mutations in the heavy chain second complementarity determining region, which is involved in antigen binding. Here we demonstrate that immunoglobulin secretion is increased up to 27-fold by incubating stably transfected PCG1-1 cells with cognate hapten p-nitrophenylphosphocholine. Secretion was unaffected by nonbinding analogs. Radiotracer and metabolic labeling experiments demonstrated specific cellular uptake of p-nitrophenylphosphocholine and increased intracellular heavy and light chain assembly. Brefeldin A inhibited hapten-mediated immunoglobulin secretion but not assembly, indicating that assembly occurs early within the biosynthetic pathway. Recovery of secretion correlated with antigen binding capacity, suggesting that the rescue mechanism involves stabilization of heavy and light chain variable domains. This model system provides the first demonstration that cognate ligands can increase intracellular assembly of functional anti-hapten antibody within mammalian cells and suggests that small molecules of appropriate specificity and affinity acting as chemical chaperones may find application for increasing or regulating immunoglobulin expression.

Purification of recombinant green fluorescent protein using chromatofocusing with a pH gradient composed of multiple stepwise fronts.

Green fluorescent protein (GFP), which fluoresces in the green region of the visible spectrum and is widely used as a reporter for gene expression and regulation, was overexpressed in the JM105 strain of Escherichia coli transformed with pBAD-GFP. A two-step chromatofocusing procedure was used to purify GFP starting from cell lysate, with each step employing a pH gradient extending from pH 5.5 to 4.0. The first chromatofocusing step was performed using a low-pressure column in which a retained stepwise pH front formed by adsorbed buffering species was used to capture GFP directly from clarified cell lysate and selectively focus it into a chromatographic band. The second step utilized a high-performance column under mass overloaded conditions where a similar pH front acted as a protein displacer and led to the formation of a highly concentrated rectangular band of GFP. The overall procedure yielded a 50-fold increase in purity, a 20-fold volume reduction, and a recovery and purity for GFP of 60% and 80%, respectively. Because the method employs a strong-base ion-exchange column packing and low-cost buffers formed with formic and acetic acids instead of the proprietary column packings and polyampholyte elution buffers more generally used for chromatofocusing, it appears to be a practical alternative for the preparative ion-exchange chromatography of GFP in particular and for the recovery of recombinant proteins from cell lysate in general. A discussion is also given concerning the choice of appropriate buffers for the rational design of pH gradients involving retained, stepwise pH fronts that span a given pH range and of the use of the fluorescence properties of GFP for flow visualization and chromatographic process development.

Cloning, expression, purification, and preliminary characterization of a putative hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803.

The genome of the unicellular cyanobacterium Synechocystis sp. PCC 6803 contains a gene (slr2097, glbN) encoding a 123 amino-acid product with sequence similarity to globins. Related proteins from cyanobacteria, ciliates, and green algae bind oxygen and have a pronounced tendency to coordinate the heme iron with two protein ligands. To study the structural and functional properties of Synechocystis sp. PCC 6803 hemoglobin, slr2097 was cloned and overexpressed in Escherichia coli. Purification of the hemoglobin was performed after addition of hemin to the clarified cell lysate. Recombinant, heme-reconstituted ferric Synechocystis sp. PCC 6803 hemoglobin was found to be a stable helical protein, soluble to concentrations higher than 500 microM. At neutral pH, it yielded an electronic absorption spectrum typical of a low-spin ferric species, with maxima at 410 and 546 nm. The proton NMR spectrum revealed sharp lines spread over a chemical shift window narrower than 40 ppm, in support of low-spin hexacoordination of the heme iron. Nuclear Overhauser effects demonstrated that the heme is inserted in the protein matrix to produce one major equilibrium form. Addition of dithionite resulted in an absorption spectrum with maxima at 426, 528, and 560 nm. This reduced form appeared capable of carbon monoxide binding. Optical data also suggested that cyanide ions could bind to the heme in the ferric state. The spectral properties of the putative Synechocystis sp. PCC 6803 hemoglobin confirmed that it can be used for further studies of an ancient hemoprotein structure.

Quantification of Endogenous Viral Polymerase, 3Dpol, in Preparations of Mengo and Encephalomyocarditis Viruses

Measurement of an antigenic response to the aphthovirus infection-associated antigen (VIA), the viral RNA polymerase 3Dpol, is frequently used as a discriminating assay for the extent of viral replication in animals. In practice, animals seropositive for VIA are assumed to have been exposed to live virus, although in fact it is suspected that endogenous 3Dpol in commercial inactivated vaccines may occasionally stimulate analogous responses and result in false-positive tests for virus exposure. Cardiovirus infections in mice produce similar anti-VIA antibodies, and in view of recently developed attenuated Mengo vaccines and live Mengo vectors, these VIA responses are also under investigation as potential correlates of vaccine efficacy. We have purified recombinant Mengo 3Dpol, developed monoclonal antibodies to the protein, and used these reagents in highly sensitive Western blot assays to quantify the levels of endogenous 3Dpol in Mengo and encephalomyocarditis virus (EMCV) preparations. The presence of 3Dpol was detected at all stages of standard vaccine purification procedures, including materials purified by CsCl. Clarified suspensions of Mengo- or encephalomyocarditis virus-infected HeLa cells were found to contain very high quantities of 3Dpol, averaging ∼1.2–1.5 μg of protein/μg of virus. Pelleting through 30% sucrose or purification by CsCl removed much of this material, but even these samples retained ∼0.2–0.4 ng of 3Dpol/μg virus. These ratios represent ∼1 3Dpol molecule/20 virus particles in the most highly purified materials and probably indicate that 3Dpol is a contaminant on the particle surface rather than an intrinsically packaged molecule. In clarified cell lysates, which are commonly used as vaccine inocula, the protein to virus ratio was ∼210:1, a level that could represent serious contamination problems for future VIA detection if such inocula are used without further purification.

Thioredoxin Domain Non-equivalence and Anti-chaperone Activity of Protein Disulfide Isomerase Mutants in Vivo

Coexpression of the enzyme, protein disulfide isomerase (PDI), has been shown to increase soluble and secreted IgG levels from baculovirus-infected insect cells (Hsu, T.-A., Watson, S., Eiden, J. J., and Betenbaugh, M. J. (1996) Protein Expression Purif. 7, 281-288). PDI is known to include catalytic active sites in two separate thioredoxin-like domains, one near the amino terminus and another near the carboxyl terminus. To examine the role of these catalytic active sites in enhancing immunoglobulin solubility, baculovirus constructs were utilized with cysteine to serine mutations at the first cysteine of one or both of the CGHC active site sequences. Trichoplusia ni insect cells were coinfected with a baculovirus vector coding for IgG in concert with either the wild-type human PDI virus, amino-terminal mutant (PDI-N), carboxyl-terminal mutant (PDI-C), or mutant with both active sites altered (PDI-NC). Western blot analysis revealed that both immunoglobulins and PDI protein were expressed in the coinfected cells. To evaluate the effect of the PDI variants on immunoglobulin solubility and secretion, the infected cells were labeled with 35S-amino-acids for different periods, and the soluble immunoglobulins were immunoprecipitated from clarified cell lysates and culture medium using anti-IgG antibodies. Only coinfections with the wild-type PDI and PDI-N mutant led to increased immunoglobulin solubility and higher IgG secretion. In contrast, infection with the PDI-C and PDI-NC variants actually lowered immunoglobulin solubility levels below those achieved with a negative control virus. Immunoprecipitation with anti-PDI antibody revealed that heterologous PDI-C and PDI-NC were insoluble, even though PDI-N and wild-type PDI protein were detected in soluble form. The capacity for PDI-N to increase immunoglobulin solubility whereas the PDI-C mutant lowered solubility indicates that the amino- and carboxyl-terminal thioredoxin domains of PDI are functionally distinct in vivo following mutations to the active site. Furthermore, mutations at the active site of the carboxyl-terminal thioredoxin domain result in PDI variants that can act as anti-chaperones of immunoglobulin solubility in vivo as has been observed in vitro for lysozyme aggregation by wild-type PDI and PDI mutants (Puig, A., and Gilbert, H. F. (1994) J. Biol. Chem. 269, 7764-7771).

Syk is required for BCR-mediated activation of p90Rsk, but not p70S6k, via a mitogen-activated protein kinase-independent pathway in B cells.

The tyrosine kinases Syk and Lyn are activated in B lymphocytes following antibody induced cross-linking of the B cell receptor for antigen (BCR). It has been suggested that activation of Syk is dependent on Lyn. We tested this hypothesis by comparing the phosphorylation and activation of several downstream effector molecules in parental DT40, DT40Syk- and DT40Lyn- B cells. The phosphorylation and activation of p90Rsk was ablated in Syk-deficient B cells but unaffected in Lyn-deficient B cells while the phosphorylation/activation of Ras GTPase activating protein (Ras GAP) and mitogen activated protein (MAP) kinase required both Syk and Lyn. Thus, these data indicate that Syk can be activated in the absence of Lyn after BCR cross-linking and results in the activation of p90Rsk via a MAP kinase-independent pathway in DT40Lyn- cells. We also demonstrated that BCR mediates the activation of p70S6k. However, activation of p70S6k in DT40Syk- and DT40Lyn- cells was comparable with that observed in parental cells. Thus, either Syk or Lyn may be sufficient for activation of p70S6k, or activation of p70S6k occurs independently of both Syk and Lyn. The kinase activity of Syk was required for the phosphorylation/activation of each of these downstream effector molecules but only the phosphorylation of Ras GAP was affected in cells expressing a mutant of Syk in which tyrosines 525 and 526 were substituted to phenlyalanines.

Purification of an L‐asparaginase—atrial natriuretic peptide fusion protein expressed in Escherichia coli

A novel fusion protein designed to facilitate protein purification was expressed in Escherichia coli and purified separately by two different chromatography methods. L-Asparaginase from Erwinia chrysanthemi is fused to the N-terminus of a model peptide, alpha-human atrial natriuretic peptide (alpha-hANP). L-Asparaginase was chosen because of its selective affinity for L-asparagine and because of its unusually high isoelectric point(8.6). A gene construction without the L-asparaginase native signal sequence caused expression at a level of 8% of total cell protein, while gene construction with the native signal sequence resulted in over five time less expression. The hybrid protein expressed without the signal sequence was purified from clarified cell lysate byeither L-asparagine affinity chromatography or cation exchange chromatography. After digestion of the fusion protein with factor Xa protease, a peptide with a molecular weight corresponding to the theoretical molecular weight of alpha-hANP was observed by coupled HPLC/mass spectrometry. (c) 1995 John Wiley & Sons Inc.

Characterization of an in vitro persistent-state measles virus infection: establishment and virological characterization of the BGM/MV cell line.

The parameters of a persistent-state measles virus infection in BGM/MV cells were examined. The BGM/MV cell line was established by cocultivation of measles virus-infected primary C3H mouse brain cells with a stable line of African green monkey kidney cells (BGM). Initially, a morphologically mixed population of cells existed:BGM-like (epithelioid) and fibroblasts. Gradually the fibroblasts were replaced by BGM-like cells, resulting in a morphologically homogeneous population. Measles cytopathic effect was noted 2 days after initiation of this culture and persisted for approximately 290 days. The time of disappearance of viral cytopathic effect corresponded to the time at which morphological homogeneity was reached. Low titers of infectious measles virus were detected in the BGM/MV culture up to 20 days postseeding; thereafter none was observed. After 440 days in culture, 100% of BGM/MV cells demonstrated intractyoplasmic measles antigen by immunofluorescence. Nuclear fluorescence was never observed. Electron microscopy revealed the presence of measles virus mucleocapsid within the almost completely filling the cytoplasm of BGM/MV cells. The plasma membrane of these cells appeared normal; no maturing or budding particles were observed. Measles virus hemagglutinin was not detected in either clarified cell lysates or in supernatant culture fluids. Cell membrane alteration by measles virus was detected in less than 1% of these cells by hemadsorption and by membrane immunofluorescence. The hemadsorption activity of the cells could be enhanced (30 to 70%) by treatment with actinomycin D or enucleation with cytochalasin B; these treatments, however, were unsuccessful in inducing detectable levels of measles hemagglutinin. Treatment of BGM/MV cells with 5-bromo-2'-deoxyuridine (BUdR) at 5 to 50 mug/ml and cytosine arabinoside at 1 to 50 mug/ml failed to enhance hemadsorption activity. Doses of 5-bromo-2'-deoxyuridine ranging from 5 to 200 mug/ml and of actinomycin D ranging from 0.1 to 10 mug/ml were ineffective in inducing the synthesis of infectious virus. Various physical methods of induction of infectious virus was also unsuccessful.