Expression of polyoma early gene products in E. coli.

The three products of the early region of polyoma virus have been cloned for expression in E. coli using the Tac promoter. Although the identical promoter and ribosome binding site are used in each final construction, the observed level of protein expression is different for each protein. While plasmids expressing wild type T antigens as well as a plasmid expressing the truncated Py-1387T middle T antigen lacking the membrane-anchoring sequence give rise to synthesis of proteins readily detectible by 35S-methionine labeling and immunoprecipitation, only small T and the middle T of Py-1387T are made in amounts sufficient for ready detection in total cell protein. Unlike middle T expressed in animal cells, middle T produced in E. coli is not detectibly phosphorylated. Further, the E. coli protein lacks tyrosine kinase activity.     

Superinfection rescue of an integrated defective polyomavirus genome.

We have characterized the viral sequences integrated in a polyomavirus-transformed mouse cell line, Py-3T3 (clone Py-6), and followed their excision and packaging upon superinfection. The polyomavirus sequences contained in Py-6 cells are present as a single insert of nonidentical tandem copies which includes, in addition to a normal middle T-antigen-coding region, some very rearranged sequences. Infection of Py-6 cells with polyomavirus strains encoding a normal large T antigen leads to the reproducible recovery in the resulting viral stock of specific defective viral genomes. The defective genomes contain a wild-type coding region for middle and small T antigens and intact viral origin and enhancer sequences. The remainder of the viral genome is rearranged or lost, so that there is no capacity to code for large T antigen or viral capsid proteins. The recovered defective sequences are also found integrated in Py-6 genomic DNA. Presumably, in infections of Py-6 cells, large T antigen, provided by the superinfecting virus, amplifies and excises the integrated viral sequences. The superinfecting helper virus must also produce viral capsids for packaging of the defective viral DNA and thus provides a means to shuttle the defective sequences from the mouse cells into other hosts, such as rat cells. In the latter host, the defective sequences are able to induce transformation.     

Genetic immunization for the recovery and purification of recombinant proteins

A system that uses genetic immunization for recombinant protein recovery and purification is described. The genetic sequence encoding a target protein is subcloned into both a eukaryotic and a prokaryotic vector. With the eukaryotic construct, a rabbit is genetically immunized and specific polyclonal antibodies to the encoded protein raised. The prokaryotic construct is used for bacterial transformation and expression of recombinant protein. Recovery and purification of target recombinant protein are obtained by passing the lysate of expressing bacteria through an immunoaffinity column prepared with the polyclonal antibodies raised in the genetically immunized animal. This method allows purification of recombinant protein without fusion tails and can be applied to purify any protein whose encoding genetic sequence is known.     

Freeze-substitution and Lowicryl HM20 embedding of fixed rat brain: suitability for immunogold ultrastructural localization of neural antigens.

We examined the suitability of freeze-substitution and Lowicryl HM20 embedding of aldehyde-fixed rat brain to localize several neural antigens at the ultrastructural level. The following rabbit polyclonal and mouse monoclonal antibodies were used: affinity-purified polyclonal immunoglobulins G raised to B-50/GAP43 (a membrane-anchored, growth-associated protein); affinity-purified polyclonal immunoglobulins G to human glial fibrillary acidic protein (GFAP; a subunit of glial filaments); a polyclonal antiserum raised to adrenocorticotropic hormone[25-39] (a neuropeptide present in dense-core granules); a polyclonal antiserum raised to myelin basic protein (a protein present in compact myelin of the central nervous system); and mouse monoclonal antibodies to synaptophysin (an integral membrane protein of small synaptic vesicles). Rat mesencephalon was fixed by perfusion with buffered 2% glutaraldehyde and 4% paraformaldehyde, cryoprotected, and frozen in liquid nitrogen. Freeze-substitution of tissue was performed with anhydrous methanol and 0.5% uranyl acetate at -90 degrees C. Semi-thin Lowicryl sections were used for light microscopic visualization of B-50 in the ventromedial mesencephalic central gray substance. The procedure preserves well the ultrastructure of this region and the immunoreactivity of the selected antigens. This study shows that dehydration by freeze-substitution, combined with Lowicryl HM20 embedding at sub-zero temperature, provides a successful method of preparation of fixed brain tissue for ultrastructural studies, allowing immunogold localization of several neural antigens by double labeling in the same section and in serial sections.     

Novel monoclonal antibodies that differentiate between the binding of pp60c-src or protein phosphatase 2A by polyomavirus middle T antigen.

Fourteen pGEX plasmids that express defined regions of polyomavirus middle T antigen in bacteria have been constructed. These polypeptides have been used to generate 18 new monoclonal antibodies directed against the unique portion of middle T and to map the approximate position of the antibody recognition sites onto the protein sequence. All of the antibodies effectively immunoprecipitate middle T and the associated 60- and 35-kDa components of protein phosphatase 2A. Four of the antibodies, however, do not react with middle T when it is bound to pp60c-src. These four probably bind to amino acids 203 to 218 of the middle T protein sequence, which are encoded by the mRNA immediately 3' to the splice junction that creates the C-terminal unique region. This suggests that additional middle T sequences are required for middle T's interaction with pp60c-src than are needed for its binding to protein phosphatase 2A. The antibodies localize this extra region and provide a means of distinguishing between these two associations.     

Production and detection of coliphage T4 endonuclease V polyclonal and monoclonal antibodies using staphylococcal protein-A hybrid proteins

To facilitate the production of antibodies against endonuclease V, a pyrimidine dimer-specific DNA glycosylase produced in bacteriophage T4-infected Escherichia coli, we constructed plasmids containing protein-A-endonuclease V fusion genes under control of the E. coli tac promoter. Induction with isopropyl-beta-D-thiogalactopyranoside produced large amounts of fusion proteins, which could easily be purified on human IgG agarose columns. The affinity-purified fusion proteins were injected into rabbits and mice to produce polyclonal and monoclonal antibodies, and also used for the screening of the monoclonal antibodies. These antibodies recognized endonuclease V on immunoblots, and also inhibited the DNA-glycosylase activity in vitro. Epitope mapping of monoclonal antibodies showed that they all (6/6) recognized determinants in the C-half of endonuclease V. A convenient way to detect primary antibodies on nitrocellulose was also developed using a crude protein extract containing protein-A-beta-galactosidase fusion protein and subsequent detection with a mixture of dyes.     

Immunological characterization of Rhizobium leguminosarum outer membrane antigens by use of polyclonal and monoclonal antibodies.

Surface antigens of Rhizobium leguminosarum biovar viciae strain 248 were characterized by using polyclonal and monoclonal antibodies. With Western immunoblotting as the criterion, an antiserum raised against living whole cells recognized mainly flagellar antigens and the O-antigen-containing part of the lipopolysaccharide (LPS). Immunization of mice with a peptidoglycan-outer membrane complex yielded eight monoclonal antibodies, of which three reacted with LPS and five reacted with various sets of outer membrane protein antigens. The observation that individual monoclonal antibodies react with sets of related proteins is discussed. Studies of the influence of calcium deficiency and LPS alterations on surface antigenicity showed that in normally grown wild-type cells, the O-antigenic side chain of LPS blocks binding of an antibody to a deeper-lying antigen. This antigen is accessible to antibodies in cells grown under calcium limitation as well as in O-antigen-lacking mutant cells. Two of the antigen groups which can be distinguished in cell envelopes of free-living bacteria were depleted in cell envelopes of isolated bacteroids, indicating that the monoclonal antibodies could be useful tools for studying the differentiation process from free-living bacteria to bacteroids.     

Differential expression of the Ebola virus GP(1,2) protein and its fragments in E. coli

Bacterial expression platforms are frequently used for the expression and production of different recombinant proteins. The full length Ebola virus (EBOV) GP(1,2) gene and subfragments of the GP(1) gene were cloned in a bacterial expression vector as a C-terminal His(6) fusion protein. Surprisingly, the full length EBOV GP(1,2) gene could not be expressed in Escherichia coli. The subfragments of GP(1) were only expressed in small amounts with the exception of one small fragment (subfragment D) which was expressed at very high levels as inclusion bodies. This was seen even in the in vitro translation system with no expression of full length GP(1,2), GP(1) subfragments A and C and low level expression of subfragment B. Only the subfragment D showed high level of expression. In E. coli (Top10), the recombinant GP(1) subfragment D protein was expressed exclusively as an insoluble approximately 25 kDa His(6) fusion protein, which is the expected size for a non-glycosylated recombinant protein. The IMAC purified and refolded non-glycosylated protein was used to immunize mice for the development of monoclonal anti-EBOV antibodies which successfully yielded several monoclonal antibodies with different specificities. The monoclonal and polyclonal antiserum derived from the animals immunized with this recombinant GP(1) subfragment D protein was found to specifically recognize the full length glycosylated EBOV GP(1,2) protein expressed in mammalian 293T cells, thus, demonstrating the immunogenicity of the recombinant subfragment.     

Properties of simian virus 40 small t antigen overproduced in bacteria

We constructed a series of bacterial plasmids which contained the Escherichia coli lac promoter fused to a simian virus 40 restriction fragment coding for small t antigen. These plasmids expressed different levels of intact viral protein depending on the length of the constructed ribosome binding site. Small t antigen synthesized by the most efficient producer, HP1, constituted 0.5 to 1% of the total cellular protein. On the basis of extensive characterization by immunoprecipitation, gel electrophoresis, isoelectric focusing, tryptic fingerprint analysis, and chromatographic properties, this plasmid-encoded protein was virtually identical to authentic simian virus 40 small t antigen. Partial purification of the HP1-encoded and authentic small t antigens revealed the presence of both monomeric and multimeric forms.     

Protein G-gold complex: comparative evaluation with protein A-gold for high-resolution immunocytochemistry

We combined the protein G-gold complex with several polyclonal and monoclonal antibodies for localization of various antigenic sites. The labelings were compared with those obtained using the protein A-gold complex. The results from either the immunodot experiment or immunoelectron microscopy have demonstrated that, for rabbit and guinea pig antibodies, both protein G-gold and protein A-gold complexes label several different specific antibodies with similar efficiency. However, with antibodies raised in goats or in mice, and particularly with mouse monoclonal antibodies, protein G-gold yielded intense and specific labeling, whereas protein A-gold yielded intense and specific labeling, whereas protein A-gold was very variable; it either gave weaker signals or failed to reveal any specific site or, as with one monoclonal, both protein G and protein A gave similar results. The higher affinity and versatility of protein G over protein A, established by the immunochemical approach, was confirmed by immunocytochemistry. Because of its enhanced reactivity with monoclonal antibodies and its broader affinity for polyclonal antibodies, protein G-gold complex appears to be a better and more versatile probe for high-resolution immunocytochemistry.     

Characterization of the interaction of polyomavirus middle T antigen with type 2A protein phosphatase.

Two cellular proteins of 36 and 63 kDa which bind the small T and middle T antigens of polyomavirus recently have been identified as the catalytic and regulatory subunits of the phosphoserine/threonine-specific type 2A protein phosphatase (PP2A). We report here the presence of phosphoseryl phosphatase activity associated with polyomavirus small T and middle T antigens in immunoprecipitates prepared from virus-infected and transformed cells. Phosphatase activity was also found associated with middle T-antigen mutants, some of which had been defined previously to associate with 36- and 63-kDa cellular proteins. Middle T-antigen-associated phosphatase activity was sensitive to okadaic acid and microcystin-LR, inhibitors of PP2A, and insensitive to inhibitor 1 or 2, orthovanadate, or EDTA. Using antiserum specific for the catalytic subunit of PP2A, we found that unlike the majority of PP2A, middle T-antigen-bound PP2A was membrane associated. However, no gross change in the amount, activity, or localization of PP2A could be attributed to middle T-antigen expression in transformed cells. Anti-PP2A antibodies coprecipitated a 63-kDa protein from normal cells and in addition coprecipitated middle T antigen, 60- and 61-kDa proteins (identified as src family members), and an 81-kDa protein from middle T-antigen-transformed cells. Furthermore, we detected protein kinase activity in PP2A immunoprecipitates and protein phosphatase activity in src immune complexes from extracts of middle T-antigen-transformed, but not normal, cells. These results reinforce the notion that at least a portion of middle T antigen bridges a protein kinase with a protein phosphatase.     

Polyoma small and middle T antigens and SV40 small t antigen form stable complexes with protein phosphatase 2A

We have purified the 36 and 63 kd cellular proteins known to associate with polyomavirus middle and small tumor (T) antigens and SV40 small t antigen. Microsequencing of the 36 kd protein indicated that it was probably identical to the catalytic subunit of protein phosphatase 2A (PP2A). Identity was confirmed by comigration on two-dimensional (2D) gels and by 2D analysis of complete chymotryptic digests. In addition, PP2A-like phosphatase activity was detected in immunoprecipitates of wild-type middle T. Immunoblotting experiments, comigration on 2D gels, and 2D analysis of limit chymotryptic digests demonstrated that the 63 kd protein, present in the middle T complex in approximately equimolar ratio to the 36 kd protein, is a known regulatory subunit of the PP2A holoenzyme. Finally, the 36 kd PP2A catalytic subunit can be immunoprecipitated by anti-pp60c-src antisera only from cells expressing wild-type middle T. These results suggest that complex formation between PP2A and T antigens may be important for T antigen-mediated transformation.     

Regions of poliovirus protein VP1 produced in Escherichia coli induce neutralizing antibodies.

Poliovirus type 1 cDNA was prepared from viral RNA encoding the VP1 capsid region of the virus by using a specific DNA primer and was cloned in Escherichia coli. DNA fragments corresponding to VP1 amino acid positions 129 to 302 (pPM5k3), 52 to 302 (pPMhae3), and 24 to 129 (pPMDxba) were incorporated into plasmid vectors designed to express Trp LE-poliovirus VP1 fusion proteins under the control of the inducible tryptophan promoter-operator system. Induction of bacterial cultures containing the plasmids resulted in the production of fusion proteins which accounted for 21% (pPMhae3), 68% (pPM5k3), and 27% (pPMDxba) of the total cell protein. The proteins were purified, and each reacted with polyclonal antibodies raised against intact virions as measured by an enzyme-linked immunosorbent assay. The sera from rabbits immunized with the bacterially produced fusion proteins pPMDxba and pPMhae3 contained poliovirus-neutralizing antibodies.     

Separation of histones from Physarum polycephalum by ion-paired, reverse-phase high-performance liquid chromatography

A rapid, one-step method for the efficient purification of murine monoclonal antibodies from tissue culture supernatants is described. This process is based on affinity chromatography on protein A-Sepharose columns. It was found that murine monoclonal antibodies raised against tick-borne encephalitis virus frequently eluted at more than one pH value and these pH values did not always correspond to those of antibodies of the same subclass from polyclonal mouse sera. The two populations of antibody molecule eluting at different pH values showed no variation in molecular weight, isoelectric profiles, specific enzyme-linked immunosorbent assay titer, or antibody subclass.     

A proteomics approach for the identification and cloning of monoclonal antibodies from serum

We describe a proteomics approach that identifies antigen-specific antibody sequences directly from circulating polyclonal antibodies in the serum of an immunized animal. The approach involves affinity purification of antibodies with high specific activity and then analyzing digested antibody fractions by nano-flow liquid chromatography coupled to tandem mass spectrometry. High-confidence peptide spectral matches of antibody variable regions are obtained by searching a reference database created by next-generation DNA sequencing of the B-cell immunoglobulin repertoire of the immunized animal. Finally, heavy and light chain sequences are paired and expressed as recombinant monoclonal antibodies. Using this technology, we isolated monoclonal antibodies for five antigens from the sera of immunized rabbits and mice. The antigen-specific activities of the monoclonal antibodies recapitulate or surpass those of the original affinity-purified polyclonal antibodies. This technology may aid the discovery and development of vaccines and antibody therapeutics, and help us gain a deeper understanding of the humoral response.     

Expression cloning of Pneumocystis carinii antigens.

We undertook expression cloning of Pneumocystis carinii antigens to overcome the difficulties encountered in purification of these antigens. Using monoclonal antibodies to the P. carinii gp120 antigen and polyclonal rabbit antiserum to rat-derived P. carinii, we have isolated cDNA clones encoding immunoreactive moieties. A cDNA clone encoding the 3' portion of a 45-55 kDa antigen of rat-derived P. carinii, was the most abundant clone isolated. The peptide encoded by this cDNA has a novel sequence with a repeated motif rich in glutamic acid residues. Affinity-purified antibodies to this peptide reacted with the 45-55 kDa band of rat-derived P. carinii. The fusion protein was recognized by serum antibodies from rats with natural exposure to P. carinii. The production of this recombinant protein should allow more detailed studies of the host-parasite relationship of this important opportunistic infection.     

Antibodies against a nonapeptide of polyomavirus middle T antigen: cross-reaction with a cellular protein(s).

Antibodies were raised against the sequence Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met -Glu, which represents a part of the middle T antigen of polyomavirus that is considered to be important in inducing the phenotype of transformed cells. The antibodies reacted with native as well as denatured middle T antigens. In addition, the antibodies immunoprecipitated a cellular protein with an apparent molecular weight of 130,000 (130K) from mouse and rat cells. In some cases, a 33K protein was also immunoprecipitated. Immunoprecipitation of middle T antigen as well as 130K and 33K proteins was blocked by the peptide. The antibodies labeled microfilaments of untransformed mouse, rat, human, and chicken cells by immunofluorescence. This labeling was also blocked by the peptide. The labeling pattern and distribution under a variety of conditions were indistinguishable from those of anti-actin antibodies, although no evidence has been obtained to indicate that the anti-peptide antibodies react with actin. The 130K protein migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis slightly slower than chicken gizzard vinculin (130K) and slightly faster than myosin light-chain kinase of chicken smooth muscle (130K). Neither of these proteins absorbed the anti-peptide antibodies. The 33K protein does not seem to be tropomyosin (32K to 40K).     

脂联素及其球状区在大肠杆菌中的可溶性表达

目的:克隆表达有生物学活性的脂联素及其球状区蛋白,并制备抗体。方法:以pQE30-adiponectin质粒为模板,PCR扩增脂联素及其球状区蛋白基因片段,插入pGEX-4T-2载体,转化大肠杆菌BL21后获得表达,用GSTrap柱亲和纯化可溶性表达的蛋白。用纯化的蛋白免疫家兔制备多抗,Westernblot鉴定抗体与人血清中脂联素的反应性。结果:PCR扩增脂联素基因片段长约710bp,脂联素球状区基因片段长约430bp。表达的GST-脂联素融合蛋白表观Mr约51000,GST-脂联素球状区融合蛋白表观Mr约42000,纯化后纯度高于90%。免疫产生的抗体与人血清中的脂联素能特异性结合。结论:表达获得的脂联素蛋白和制备的抗体为脂联素的检测及对其功能的研究奠定了基础。