Characterization of the chromatin acceptor sites for the avian oviduct progesterone receptor using monoclonal antibodies

Monoclonal antibodies (MAb) against the chromatin acceptor sites for the avian oviduct progesterone receptor were prepared with highly purified hen oviduct acceptor proteins reconstituted to hen DNA. Addition of the MAbs to a cell-free assay blocked progesterone receptor from chick oviduct (PRov) binding to native-like acceptor sites on nucleoacidic protein (NAP) representing a partially deproteinized chromatin, which has been shown to be enriched in these binding sites. However, the antibodies do not block PRov binding to pure DNA, nor do they affect the receptor itself. Estrogen receptor binding to NAP was not inhibited, supporting a receptor specificity of the PRov acceptor sites as reported previously from direct competition studies. These data support earlier studies showing that (1) the reconstituted PRov acceptor sites resemble the native sites, (2) the acceptor sites are receptor specific, and (3) the PRov binding sites of NAP are different from those of pure DNA. While some animal-species specificity in the PRov binding inhibition was observed, no tissue specificity was seen. Direct binding of the antibodies to native acceptor sites was demonstrated in an enzyme-linked immunosorbent assay (ELISA) system. The antibodies showed little recognition of free acceptor protein or DNA alone, indicating specificity for the protein-DNA complex. A partial evolutionary conservation of the nuclear acceptor sites for PRov was shown by the fact that about 50% of the inhibition seen with hen NAP was obtained with NAPs from several other species, and this partial cross-reactivity of the MAbs with the same NAPs from other animal species was also seen in the ELISA.     

Characterization of a purified chromatin acceptor protein (receptor binding factor 1) for the avian oviduct progesterone receptor

The specific, high-affinity binding of the avian oviduct progesterone receptor (PR) with target-cell nuclei and chromatin has been shown to involve DNA complexed with specific chromatin acceptor proteins. One of these chromatin acceptor proteins has been partially purified and found to be a small hydrophobic protein with a broad pI of 5.0-6.0 [Goldberger, A., & Spelsberg, T. C., (1988) Biochemistry 27, 2103-2109]. This paper describes the final purification over 100,000-fold to apparent homogeneity of this candidate PR acceptor protein, termed the receptor binding factor 1 (RBF-1). When the avian genomic DNA is bound by RBF-1, saturable, high-affinity (KD approximately 2 x 10(-9) M) binding sites for PR are generated. RBF-1 has a unique, hydrophobic N-terminal sequence. The PR binding to the RBF-1-DNA complexes is shown to be dependent on an intact activated PR with which excess nonradiolabeled PR can compete. By use of a new, highly specific monoclonal antibody (mAb) to the RBF-1 with Western immunoblotting, RBF-1 was shown to be localized in the nucleus and to be tissue and species specific. Selective removal of the chromatin proteins containing RBF-1 results in the loss of the highest affinity class of PR binding sites. A second class of residual PR binding sites remains in the nucleoacidic protein (NAP), a complex of proteins more tightly bound to the DNA. This class of PR binding activity has been classified as the RBF-2. The RBF-1 is estimated to be 0.03% of the total chromatin protein with about 1.2 x 10(5) molecules/diploid cell.     

Enrichment of a second class of native acceptor sites for the avian oviduct progesterone receptor as intact chromatin fragments

Several classes of specific progesterone receptor (PR) nuclear binding sites (acceptor sites) have previously been identified in avian oviduct chromatin on the basis of different binding affinities. Recently, two classes of acceptor proteins (AP) that are associated with these binding sites in the avian oviduct have been identified. These APs were termed receptor binding factors (RBF-1 and -2), and one (RBF-1) has been purified [Schuchard et al. (1991) Biochemistry 30, 4535-4542]. The RBF-1 is associated with the highest affinity class of sites in the intact chromatin, and the RBF-2 is associated with the second highest affinity class of sites. The PR binding sites and their associated RBF-2 protein remain with the residual chromatin fraction following extraction by 4 M Gdn-HCl. This Gdn-HCl-treated chromatin has been termed nucleoacidic protein (NAP). This paper describes the 200-fold enrichment of the native RBF-2 class of PR acceptor sites beginning with the DNase I digestion of NAP to obtain DNase-resistant fragment (NAPf) containing approximately 150 bp of DNA. The PR binding sites are further enriched by high-performance or fast protein liquid chromatography and chromatofocusing. Anti-RBF-1/RBF-2 protein antibodies identify antigens that coelute with the PR binding activity. Hybridization analysis of the DNAf from the enriched NAPf demonstrates sequence homologies with the nuclear matrix DNA as well as with genomic sequences of the rapid steroid responding nuclear protooncogenes c-myc and c-jun. However, comparative analyses of the whole genomic DNA with the nuclear matrix DNA indicate that the RBF-2 (NAPf) is largely nonnuclear matrix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstitution of nativelike nuclear acceptor sites of the avian oviduct progesterone receptor: evidence for involvement of specific chromatin proteins and specific DNA sequences

A specific fraction of avian oviduct chromosomal proteins can be reannealed to pure avian DNA to reconstitute nativelike specific nuclear binding sites (acceptor sites) for the oviduct progesterone receptor (PR). These specific nuclear binding sites represent the difference between the binding to the reconstituted NAP and that to pure DNA. The specific fraction of chromatin protein which contains the acceptor activity, fraction CP-3, is very tightly bound to hen DNA in a complex termed nucleoacidic protein (NAP). Removal of the CP-3 fraction from NAP results in a loss of specific PR binding sites. Resins containing chromatin adsorbed to hydroxylapatite are used as a rapid method to isolate the CP-3 fraction. Reconstitution of the CP-3 fraction to DNA by the described method involving a regressing gradient of 6-0 M guanidine hydrochloride (Gdn-HCl) results in a reconstituted NAP which displays specific PR binding sites identical with those in native (undissociated) NAP and whole chromatin. Optimal conditions and potential problems for reconstituting these nucleoproteins are described. Only partially purified receptor preparations were used in these cell-free binding analyses since they have been shown to bind with similar properties and patterns as the nuclear binding in vivo. Therefore, the binding of PR to the reconstituted NAPs was demonstrated to be receptor dependent, saturable, and of high affinity. Further, the pattern of binding to the reconstituted sites mimics those which are observed in vivo. Thus, nonfunctional receptors that cannot translocate and bind to the nuclear acceptor sites in vivo also failed to bind to the acceptor sites on the reconstituted NAPs generated by the acceptor proteins. In contrast, the binding to pure DNA does not reflect these receptor differences in receptor bindings. Specific binding of PR to reconstituted NAP can be reversed by again removing the protein fraction. Moreover, the specific binding can be destroyed by proteases and protected by protease inhibitors, indicating that acceptor activity is proteinaceous in nature. The reconstitution of the activity is both a concentration-dependent and time-dependent process. During the reconstitution, acceptor activity appears to reconstitute on the DNA when the Gdn-HCl concentration reaches 2.0 M. By use of the reconstitution method as an assay for acceptor activity, the activity in the CP-3 fraction was shown by molecular sieve chromatography to elute in a relatively broad molecular weight range between 13 000 and 25 000. The activity also focuses in isoelectric focusing resins with apparent pI's of 5.2 and 6.4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification of a nuclear protein (Receptor Binding Factor-1) associated with the chromatin acceptor sites for the avian oviduct progesterone receptor

The specific high affinity binding of the avian oviduct progesterone receptor (PR) to target cell nuclei and chromatin has been shown to involve DNA complexed with specific chromatin acceptor proteins. One of these chromatin acceptor proteins has been partially purified and found to be a small hydrophobic protein with a broad pI of 5.0–6.0 [Goldberger and Spelsberg (1988),Biochem. 27, 2103–2109]. Using western immunoblots with anti-RBF-1 polyclonal antibodies to monitor the purification, a 10 kD candidate acceptor protein, termed the Receptor Binding Factor-1 (RBF-1), has been purified to apparent homogeneity. RBF-1 has an amino acid composition consistent with a hydrophobic protein having an acidic pI and a unique N-terminal sequence. Two-dimensional polyacrylamide gel electrophoresis and high-performance capillary electrophoresis support the purity of a protein 10 kD in size, having an acidic pI, but with evidence of several differently charged isoforms. Phosphatase treatment provides evidence that charge heterogeneity may result from variable phosphorylation states. A role of this factor as a candidate acceptor protein in the chromatin acceptor sites for the avian oviduct PR is proposed.     

An automatic multidimensional chromatography system for purification of human uterine progesterone receptor and induction of polyclonal antibodies

This paper reports on the synthesis of Org2058-bonded microparticulate silicas and their use in affinity chromatography as the first step for the purification of human progesterone receptor. The development of microprocessor-controlled instruments allows all the various steps to be performed automatically. The various steps used for the purification of human progesterone receptor were carried out with the FPLC system: affinity chromatography, desalting of eluate on Sephadex G-25, anion-exchange chromatography using a Mono Q column. With this procedure the receptor was purified approx. 10,000-fold within 24 h. The yield of receptor was generally 85-95%. Investigations with induced anti-progesterone receptor antibodies obtained after the fourth immunization show their immunoreactive behaviour towards progesterone receptor in crude cytosol, which was proved by sucrose density gradient centrifugation and by gel filtration on the FPLC system using a Sepharose 12 column. This implies that progesterone receptor was efficiently purified by our purification procedure.     

Rat antibodies as probes for the characterization of progesterone receptor A and B proteins from laying hen oviduct cytosol

The chicken oviduct contains two different hormone binding forms of the progesterone receptor, A and B. We have prepared rat antisera against both forms of the receptor partially purified from laying hen oviduct. The anti-progesterone receptor A antiserum reacts with both receptor forms on Western blots, while the anti-progesterone receptor B antiserum reacts mainly with the B form. Both antisera also react with the native progesterone receptor proteins as shown by sedimentation analysis of the antibody-receptor complexes. Receptors A and B are recognized on Western blots of total protein from dissolved tissue, indicating that both forms are likely to be physiological components. Epitope mapping experiments show that immunogenicity of both receptor molecules is restricted to structurally related protein domains of 28 kDa in receptor A and of 52 kDa in receptor B.     

Antibodies against highly purified B-subunit of the chick oviduct progesterone receptor

A rabbit was immunized with the highly purified B-subunit (110kDa) (20 to 50 micrograms per injection) of the chick oviduct progesterone receptor (PR). Specific antibodies (IgG-RB) were observed 2 weeks after the first booster injection and high antibody titers in the serum were found after the second and third booster injections (with Kdeq of interaction integral of 2 nM). IgG-RB were tested by immunoprecipitation, immunoblotting, density gradient ultracentrifugation and protein A-sepharose assay methods. They recognized not only the B-subunit but also the A-subunit (79K), the nuclear PR, the mero-receptor (proteolytic cleavage product) and the "non-activated" molybdate-stabilized "8S" PR. However, IgG-RB did not interact with the 90K non hormone-binding component of this 8S-PR. IgG-RB did not affect the binding of the hormone to PR, whether incubated with the receptor before or after labelling with tritiated progesterone. They did not cross-react with glucocorticosteroid receptor of the chick oviduct. Weak interaction was observed with estrogen receptor of the chick oviduct and with KC1 activated "4S" forms of the rabbit and human uterus PR.     

双峰驼血清IgG亚型的分离纯化及多克隆抗体的制备

双峰驼IgG亚型包含IgG1、IgG2和IgG3,其中IgG2和IgG3为重链抗体,在结构上与IgG1存在显著差异。为获取双峰驼血清中的IgG1、IgG2和IgG3,并分析其抗原特异性和抗体特异性,本文交替使用Protein A和Protein G亲和层析柱,对其分离纯化,并通过聚丙烯酰胺凝胶电泳进行鉴定;之后分别制备兔抗双峰驼IgG1、IgG2和IgG3的多克隆抗体,通过ELISA对制备的多克隆抗体的效价进行测定;最后应用Western blot评估这三个亚型多克隆抗体的特异性,进而对双峰驼血清中IgG1、IgG2和IgG3的抗原特异性进行分析。结果表明,应用Protein A和Protein G亲和层析柱成功分离纯化出双峰驼血清中的IgG1、IgG2和IgG3;并制备兔抗双峰驼IgG1、IgG2和IgG3的多克隆抗体效价均在1∶10000以上,并且所获得的多克隆抗体分别与IgG1、IgG2和IgG3之间均存在交叉反应,但兔抗双峰驼IgG1多克隆抗体较其它两个亚型多克隆抗体特异性低。结果证明,双峰驼IgG1、IgG2和IgG3均具有良好的免疫原性,三者结构虽存在显著差异,但其抗原特性类似。     

Interaction of murine progesterone receptors with specific monoclonal antibodies to the avian progesterone receptor

Monoclonal antibodies raised against purified chicken progesterone receptor (PgR) have been described and characterized recently. In this study we have screened these antibodies for cross-reactivity with murine PgR. Of the six anti-PgR antibodies tested, one (alpha PR6) precipitates murine PgR in an assay using protein A-sepharose as an absorbent for the antibody. The antibody is specific for PgR and does not react with the estrogen receptor or the glucocorticoid receptor in the same cytosol. In immunoblot experiments, both alpha PR6 and alpha PR11 recognize a 115,000 Da protein, however, alpha PR11 gives a weaker signal than alpha PR6. In photoaffinity labeling experiments, a 115,000 Da and an 83,000 Da protein covalently bind tritiated R5020 in a receptor-specific way. We conclude that the alpha PR6 antibody can be used as a tool to study the structure and function of the murine PgR.     

诺如病毒NV衣壳蛋白VP1表达纯化及多克隆抗体的制备

目的 获得纯化的诺如病毒（NV）衣壳蛋白VP1,免疫动物制备多克隆抗体。方法 提取粪便样品中诺如病毒RNA,逆转录得到cDNA文库,通过PCR扩增获取VP1基因序列,构建到大肠埃希菌原核表达系统中诱导表达重组VP1蛋白。使用镍柱亲和层析法对重组蛋白进行纯化,十二烷基磺酸钠‒聚丙烯酰胺凝胶电泳（SDS-PAGE）和考马斯亮蓝法（BSA）对重组蛋白的纯度与浓度进行分析,以重组的VP1蛋白为抗原,免疫雄性SPF级SD大鼠获得多抗血清,用ELISA测定抗体效价、Western blot检测抗体特异性。结果 成功地构建出重组表达载体VP1-pET28a,并将其在大肠埃希菌BL21（DE3）中稳定地表达诱导重组蛋白。ELISA测其多抗血清的平均效价为1∶200000,Western blot检测抗体在原核和真核特异性很高。结论 本实验成功地利用原核表达系统表达诺如病毒衣壳蛋白VP1,为进一步研究诺如病毒的诊断和疫苗开发提供了条件。     

Binding of heat shock proteins to the avian progesterone receptor.

The protein composition of the avian progesterone receptor was analyzed by immune isolation of receptor complexes and gel electrophoresis of the isolated proteins. Nonactivated cytosol receptor was isolated in association with the 90-kilodalton (kDa) heat shock protein, hsp90, as has been described previously. A 70-kDa protein was also observed and was shown by Western immunoblotting to react with an antibody specific to the 70-kDa heat shock protein. Thus, two progesterone receptor-associated proteins are identical, or closely related, to heat shock proteins. When the two progesterone receptor species, A and B, were isolated separately in the absence of hormone, both were obtained in association with hsp90 and the 70-kDa protein. However, activated receptor isolated from oviduct nuclear extracts was associated with the 70-kDa protein, but not with hsp90. A hormone-dependent dissociation of hsp90 from the cytosolic form of the receptor complex was observed within the first hour of in vivo progesterone treatment, which could explain the lack of hsp90 in nuclear receptor complexes. In a cell-free system, hsp90 binding to receptor was stabilized by molybdate but disrupted by high salt. These treatments, however, did not alter the binding of the 70-kDa protein to receptor. Association of the 70-kDa protein with the receptor could be disrupted by the addition of ATP at elevated temperatures (23 degrees C). The receptor-associated 70-kDa protein is an ATP-binding protein, as demonstrated by its affinity labeling with azido[32P]ATP. These results indicate that the two receptor-associated proteins interact with the progesterone receptor by different mechanisms and that they are likely to affect the structure or function of the receptor in different ways.     

Nuclear matrix localization and specific matrix DNA binding by receptor binding factor 1 of the avian oviduct progesterone receptor

A chromatin acceptor protein for the avian oviduct progesterone receptor (PR), termed receptor binding factor 1 (RBF-1), has recently been shown to (1) be a component of the nuclear binding sites (acceptor sites) for PR and (2) generate high-affinity binding sites (termed the RBF-1 class of sites) on avian genomic DNA [Schuchard et al. (1991) Biochemistry 30, 4535-4542]. A second class of sites and its associated protein (termed RBF-2) were also identified. This paper demonstrates that RBF-1 and also the PR nuclear binding sites are localized in the oviduct nuclear matrix. RBF-1 is found in abundance in the nuclear matrix of liver but only in traces in the nuclear matrix of spleen. Extraction of the nuclear matrix with 4.0 M Gdn-HCl results in the complete removal of RBF-1 as occurs with whole chromatin. Interestingly, a second class of specific PR binding, termed RBF-2, remains on the nuclear matrix after the removal of all RBF-1. Southern blot analysis indicates that the nuclear matrix DNA contains sequences homologous with the 5'-flanking domains of the rapidly steroid regulated c-myc and c-jun protooncogenes and the beta-actin gene, but not genomic sequences of the late sex steroid regulated gene, ovalbumin, or the alpha-actin gene. A specific, small region in the 5'-flanking domain of the c-myc gene appears to be associated with the nuclear matrix. Southwestern blot analysis using partially purified RBF-1 shows a marked affinity and specificity of the RBF-1 for the nuclear matrix DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

A polyclonal antiserum against the rabbit progesterone receptor recognizes the human receptor: biochemical characterization

Polyclonal antiserum was generated in guinea pigs immunized with the 116,000 Mr rabbit uterine progesterone receptor (PR). The PR antigen was partially purified by DEAE-cellulose chromatography and preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and the 116,000 Mr band excised and injected into guinea pigs. The antiserum recognized on protein blots rabbit uterine PR of Mr 116,000 and 81,000. The antiserum was judged to be specific for PR from normal and malignant human tissues as determined by sedimentation shift on sucrose gradients, immunoprecipitation studies, protein blotting, and fluorographic analysis using photolabelled samples. Comparison of protein blots probed with this polyclonal antiserum or with a recently obtained monoclonal antibody to human PR indicated that similar PR structures were recognized in rabbit and human samples by both antisera. Characterization of the polyclonal antiserum has demonstrated its suitability for investigating the immunolocalization or PR in normal and malignant human tissues as well as the receptor structure detected on protein blots.     

重组丙型肝炎病毒核心蛋白的原核表达、纯化和抗体制备

本文旨在获得纯化丙型肝炎病毒(HCV)核心蛋白(HCV-C)及抗HCV-C多克隆抗体,为深入研究HCV-C与肝细胞相互作用的分子机制奠定基础。首先以HCV1b亚型HC-J4-91全基因组质粒为模板,聚合酶链反应(PCR)扩增HCV-C基因,构建重组质粒pQE31-HCV-C。融合蛋白经原核表达、纯化后,免疫BALB/c小鼠,制备抗HCV-C多克隆抗体。利用酶联免疫吸附试验(ELISA)检测抗体效价,蛋白免疫印迹(Westernblot)和间接免疫荧光染色鉴定抗体特异性。结果显示,表达HCV-C的原核表达质粒pQE31-HCV-C构建正确,获得相对分子质量约22000的纯化融合蛋白。ELISA检测重组蛋白免疫小鼠的抗血清效价达1:12800。结果显示,自制的抗HCV-C多克隆抗体能特异性识别HCV-C。本研究获得了纯度较好、原核表达的HCV-C,并成功制备了抗HCV-C多克隆抗体,为深入研究HCV-C的致病机制提供了有实用价值的研究工具。     

Chick oviduct glucocorticosteroid receptor. Specific binding of the synthetic steroid RU 486 and immunological studies with antibodies to chick oviduct progesterone receptor

The glucocorticosteroid receptor (GR) has been studied in oviduct cytosol prepared from estrogen-primed, 4-week-withdrawn chicken. The equilibrium dissociation constant was 6 nM for dexamethasone, and 18 300 receptor sites/cell were measured assuming that all cells contain identical concentrations of GR. Dexamethasone, used in most studies investigating glucocorticosteroid action, was found not to be the best GR ligand. The affinities of several natural and synthetic glucocorticosteroids for GR increased in the following order: cortisol less than deoxycorticosterone less than dexamethasone less than corticosterone less than triamcinolone acetonide. The synthetic steroid RU 486 was the most specific ligand of GR (its affinity was approximately equal to 10-fold higher than that of triamcinolone acetonide), while it did not bind either to plasma transcortin (which binds dexamethasone nor, surprisingly, to progesterone receptor (PR), contrary to what occurs in mammalian species. The molybdate-stabilized, 8-S form of GR was prepared from withdrawn chick oviduct, whole chick embryo or cultured chick embryo fibroblasts (which do not contain PR), and was labeled with either [3H]dexamethasone or [3H]RU 486. The sedimentation coefficient of radioactive ligand--8-S GR complexes was shifted towards heavier forms after incubation with polyclonal (IgG-G3) or monoclonal (BF4) antibodies generated against the molybdate-stabilized, 8-S form of the chick oviduct PR. Since neither IgG-G3 nor BF4 interacted with the steroid binding 4-S form of GR, it is suggested that these antibodies recognized a non-steroid binding protein common to molybdate-stabilized, 8-S forms of GR and PR.