Two cytoplasmic candidates for immunophilin action are revealed by affinity for a new cyclophilin: one in the presence and one in the absence of CsA.

We report the cloning and characterization of a new binding protein for the immunosuppressive drug cyclosporin A (CsA). This new cyclophilin, cyclophilin C (cyp C), shows extensive homology with all previously identified cyclophilins. Cyp C mRNA is expressed in a restricted subset of tissues relative to cyclophilins A and B, but is present in those tissues reported to be most affected by CsA therapy. A cyp C fusion protein has peptidyl-prolyl isomerase activity, and CsA inhibits this activity. Using the cyp C fusion protein as an affinity ligand to probe cellular extracts, we find that the cyp C fusion protein binds specifically to a 77 kd protein in the absence of CsA, while in the presence of CsA it instead binds specifically to a 55 kd protein. We propose that the p77 is involved in cyp C native function and that the p55 is involved in signal transduction events blocked by treatment with immunosuppressive levels of CsA.     

Identification of actin and HSP 70 as cyclosporin A binding proteins by photoaffinity labeling and fluorescence displacement assays.

A novel family of cyclosporin A (CsA) binding proteins was identified by using the biologically active, radioiodinated photoaffinity probe [D-Lys-N epsilon-(4-azido-3-[125I]iodophenyl)propionyl)]8-CsA. In addition to cyclophilin, proteins with molecular masses of 43 kDa and approximately 50-55 kDa were labeled in Jurkat extracts and bovine calf thymus. Sequence analysis of the 43-kDa protein purified from calf thymus and subsequent Western analysis of CsA affinity-purified material from Jurkat extracts identified the 43-kDa component as actin. [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA, a fluorescent analogue of CsA, was prepared and used to measure the binding constants of cyclosporin derivatives to actin by means of a new fluorescence displacement assay. [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA and [N delta-t-butoxycarbonyl diaminobutyryl)]8-CsA bind to bovine actin at physiologically relevant concentrations, with dissociation constants of 60 +/- 33 and 570 +/- 380 nM, respectively. Because the ATPase fragment of heat shock cognate 70 (HSC 70) is structurally related to actin, the yeast homologue SSA1 was tested and found to be radiolabeled by the cyclosporin A photoaffinity reagent. The binding constant for [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA to SSA1 was determined and is 53 +/- 48 nM. These results indicate that actin and the 70-kDa heat shock protein family contain a structurally related domain for binding of cyclosporin A-related peptides.     

Host-cell cyclophilin is important for the intracellular replication of Leishmania major

The antiparasitic effects of cyclosporin A were examined in leishmanial infection by analysing the role of CsA-binding proteins (cyclophilins) in the host–parasite interaction. We hypothesized that the leishmanicidal effects of CsA on Leishmania major infected macrophages might be mediated through a cyclophilin of either the parasite or the host cell. Two cyclophilins (20 and 22 kDa) were purified from L. major parasites and N-terminally sequenced. Although enzyme activity of these cyclophilins was inhibited by CsA, pretreatment of L. major parasites with CsA did not result in reduction of a subsequent macrophage infection, arguing against a role of L. major cyclophilins as infectivity potentiators. However, host-cell cyclophilin A (CypA) was found to be critically involved in the intracellular replication of L. major parasites in murine macrophages. An antisense oligonucleotide to murine CypA was constructed and added to cultures of peritoneal macrophages prior to infection with L. major parasites. This treatment strongly reduced the expression of CypA in macrophages and resulted in the inhibition of the intracellular replication of L. major amastigotes. These data indicate that interaction of amastigotes with host-cell cyclophilin is an important part of the intracellular replication machinery of L. major and define, for the first time, a direct involvement of a cyclophilin in the survival strategies of an intracellular parasite.     

Characterisation of a cyclophilin isoform in Trypanosoma cruzi

The immunosuppressive drug cyclosporin A (CsA) has shown antiparasitic activity against several protozoans and helminths, when complexed to proteins called cyclophilins (CyPs). In this paper, the molecular characterisation of one member of the CyP family in Trypanosoma cruzi is reported. TcCyP19 gene proved to be highly conserved compared to CyPs from other organisms and was highly homologous to a Trypanosoma brucei brucei CyPA. This gene was expressed in Escherichia coli and the purified recombinant protein exhibited a peptidyl prolyl cis-trans isomerase activity that was inhibited by CsA (IC(50) = 18.4 + /-0.8 nM). The TcCyP19 gene was located on two chromosomal bands in T. cruzi CL Brener clone.     

Isolation of a unique membrane protein from Naegleria fowleri

Naegleria fowleri, an amoeboflagellate, is the causative agent of Primary Amoebic Meningoencephalitis, a fulminating disease of the central nervous system. In order to elucidate the mechanisms of pathogenicity of this amoeba, a cDNA expression library was prepared from N. fowleri RNA. A specific protein was found to be expressed from a cDNA clone designated Mp2CL5. Northern blot analysis showed that the Mp2CL5 mRNA was expressed in pathogenic N. fowleri but was not expressed in non-pathogenic Naegleria species nor in Acanthamoeba. Western blot analysis using anti-N. fowleri antiserum demonstrated that IPTG-induced Escherichia coli Mp2CL5 expressed a 23-kDa recombinant protein. The Mp2CL5 recombinant protein was histidine-tagged and purified to homogeneity from E. coli. A polyclonal rabbit antiserum was prepared against the purified Mp2CL5 recombinant protein. This antibody was used to further characterize the Mp2CL5 native protein expressed by N. fowleri. Western blot analysis in conjunction with immunofluorescence microscopy demonstrated the presence of a native protein of 17 kDa on the plasma membrane of N. fowleri trophozoites. The native N. fowleri protein was expressed in the logarithmic phase of trophozoite growth and the production of this protein increased through the stationary phase of growth. Studies are in progress to examine further its role as a virulence factor.     

Kidney androgen-regulated protein interacts with cyclophilin B and reduces cyclosporine A-mediated toxicity in proximal tubule cells

The gene for kidney androgen-regulated protein (KAP) is the most abundant and specific gene expressed in mouse kidney proximal tubule cells, where it is tightly regulated by steroid and thyroid hormones in different tubule segments. Despite the cell-specific expression, strict regulatory mechanisms, and relative abundance, nothing is known of the function of its encoded protein, which does not exhibit known structural or functional domains, or homologies with other sequences in the data bases. We raised monoclonal antibodies against KAP, which specifically recognize a protein with an apparent molecular mass of 20 kDa in crude kidney homogenates, the distribution and regulation of which parallel that of its mRNA. To gain insight into its function, we performed a yeast two hybrid screen and determined that KAP specifically interacts with cyclophilin B. Furthermore, cyclosporine A (CsA)-treated mice exhibited a significant decrease in KAP levels, and tetracycline-controlled overexpression of KAP in stably transfected proximal tubule cells significantly decreased the toxic effects of CsA. Taken together, these results indicate a functional relationship among KAP-, cyclophilin B-, and CsA-mediated nephrotoxicity and suggest an important role of KAP in renal physiology, providing new data on the molecular mechanisms implied in the toxic effects of CsA.     

Immunological comparison of native and recombinant egg allergen, ovalbumin, expressed in Escherichia coli

Chicken ovalbumin is one of the major egg white allergens which causes IgE-mediated food hypersensitivity. A gene encoding for chicken ovalbumin (Gad dI) was isolated from chicken oviduct by PCR amplification and was cloned under the control of T5 promoter fused with a six-histidine tag at the N-terminal end. Escherichia coli harbouring this construct expressed high quantities of the recombinant protein in the form of soluble fraction. The protein was purified using affinity chromatography on a Ni(2+)-nitrilotriacetic acid agarose column and was further purified to homogeneity by ion exchange chromatography. Homogeneity was confirmed through SDS-PAGE, Western blot and secondary conformation analysis. The reactivity of the recombinant and native protein was tested against six egg allergic human patient's sera and the IgE and IgG binding activity was tested using both Western blot and ELISA. When compared to native ovalbumin, the recombinant protein had similar binding activity in immunoblotting, but slightly increased activity by ELISA. Circular dichroism revealed that the recombinant protein had a slightly less compact structure than the native form. Both antigens exhibited a similar immunogenicity in mice.     

兔抗人热激蛋白70样蛋白1多克隆抗体的制备与初步鉴定

目的：制备兔抗人热激蛋白70样蛋白1（HSP70L1）的多克隆抗体并进行初步鉴定。方法：在大肠杆菌中重组表达融合蛋白GST-HSP70L1和His-HSP70L1并纯化;将GST-HSP70L1融合蛋白用于免疫新西兰大耳白兔获得多克隆抗体,用His-HSP70L1对抗血清进行分离纯化,得到抗HSP70L1多克隆抗体,用Western印迹、免疫沉淀对其进行初步鉴定。结果：获得了高表达的GST-HSP70L1和His-HSP70L1重组融合蛋白;纯化获得抗HSP70L1抗体,此抗体可用于Western印迹和免疫沉淀实验。结论：获得了兔抗人HSP70L1的多克隆抗体,为进一步研究HSP70L1的生物功能提供了有用的工具。     

Immunocytochemical demonstration of erythropoietin in hypoxic human hepatoma cultures

The possibility of demonstrating erythropoietin at the light microscopic level was examined in homogeneous cultures of the erythropoietin-producing human hepatoma cell lines HepG2 and Hep3B. Immunoperoxidase staining was applied in combination with several mono- and polyclonal antibodies. Sufficiently strong colour responses were obtained with all three polyclonal antibodies and with one of three monoclonal antibodies raised against recombinant human erythropoietin. The staining intensity was increased in hypoxic versus non-hypoxic hepatoma cultures. Intracellular erythropoietin immunoreactivity was confirmed by Western blot analysis of HepG2 extracts. The effect of oxygen supply on erythropoietin gene expression was confirmed by competitive polymerase chain reaction of erythropoietin mRNA and by radioimmunoassay of secreted erythropoietin.     

A single Trp121 to Ala121 mutation in human cyclophilin alters cyclosporin A affinity and peptidyl-prolyl isomerase activity

Fluorescence and NMR spectral data have suggested an interaction between the single tryptophan in cyclophilin (CyP) and its high affinity ligand cyclosporin A (CsA). To study this interaction, a site mutation of Trp121 to Ala was introduced into human cyclophilin (CyP) and the encoded protein was expressed in E. coli. The Ala121 mutant was shown to catalyze the peptidyl-prolyl cis-trans isomerase (rotomase) reaction with several peptide substrates, albeit at less than ten percent the rate of the purified recombinant human CyP. Values for the apparent inhibition constant (Ki,app) of cyclosporin A with the human CyP and the Ala121 mutant were determined to be 1.6 +/- 0.4 nM and 640 +/- 90 nM, respectively by tight-binding inhibition analysis. The greater loss of affinity for CsA binding (400-fold) than for rotomase catalysis (20 fold) suggests that the catalytic and CsA binding properties associated with CyP can be decoupled as has been observed with an homologous protein found in E. coli (Liu, J. & Walsh, C.T. (1990) Proc. Natl. Acad. Sci. USA 87, 4028-4032).     

Production of Polyclonal Antibodies to the Recombinant Potato virus M (PVM) Non‐structural Triple Gene Block Protein 1 and Coat Protein

The genes encoding the coat protein (CP) and triple gene block protein 1 (TGBp1) of Potato virus M (PVM) were cloned into expression vector pET‐45b(+) (N‐terminal 6xHis tag) and expressed in E. coli Rosetta gami‐2(DE3). The purified recombinant antigens were used for raising polyclonal antibodies. The antibodies against recombinant CP were successfully used in Western blot analysis, plate‐trapped ELISA and DAS‐ELISA as a coating for PVM detection in infected potato leaf samples. The antibodies against recombinant non‐structural protein detected the TGBp1 only in Western blot analysis. This is the first report of the production of polyclonal antibodies against recombinant coat protein and TGBp1 of PVM and their use for detecting the virus.     

Hyoscyamine 6 beta-hydroxylase, an enzyme involved in tropane alkaloid biosynthesis, is localized at the pericycle of the root

Hyoscyamine 6 beta-hydroxylase (H6H; EC 1.14.11.11) catalyzes the first reaction in the biosynthetic pathway from hyoscyamine to scopolamine in several solanaceous plants. Four monoclonal antibodies were raised against H6H purified from cultured roots of Hyoscyamus niger. The IgG1 antibody mAb5 inhibited H6H activities present in cell-free extracts of H. niger roots and specifically recognized 38-40-kDa proteins from six different scopolamine-producing plant species in Western blot analysis after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The other three monoclonal antibodies all recognized SDS-denatured H6H protein from Hyoscyamus species, but did not bind to native H6H. Western blot analysis of protein extracts from various tissues of H. niger using these antibodies showed that H6H is abundant in cultured roots, present in plant roots, but absent in leaf, stem, calyx, cultured cells, and cultured shoots. Immunohistochemical studies using monoclonal antibody and immunogold-silver enhancement detected H6H only in the pericycle cells of the young root in several scopolamine-producing plants. Mature roots that underwent secondary growth and lacked the pericycle did not react with the antibody. This pericycle-specific localization of scopolamine biosynthesis provides an anatomical explanation for the tissue-specific biosynthesis of tropane alkaloids and may be important for translocation of tropane alkaloids from the root to the aerial parts.     

The antigenic and catalytically active formamidase of Paracoccidioides brasiliensis: protein characterization, cDNA and gene cloning, heterologous expression and functional analysis of the recombinant protein

Paracoccidioides brasiliensis is a well-characterized pathogen of humans. To identify proteins involved in the fungus-host interaction, P. brasiliensis yeast proteins were separated by liquid isoelectric focusing, and fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Immunoreactive bands were detected with pooled sera of patients with P. brasiliensis infection. A protein species with a molecular mass of 45 kDa was subsequently purified to homogeneity by preparative gel electrophoresis. The amino acid sequence of four endoproteinase Lys-C-digested peptides indicated that the protein was a formamidase (FMD) (E.C. 3.5.1.49) of P. brasiliensis. The complete cDNA and a genomic clone (Pbfmd) encoding the isolated FMD were isolated. An open reading frame predicted a 415-amino acid protein. The sequence contained each of the peptide sequences obtained from amino acid sequencing. The Pbfmd gene contained five exons interrupted by four introns. Northern and Southern blot analysis suggested that there is one copy of the gene in P. brasiliensis and that it is preferentially expressed in mycelium. The complete coding cDNA was expressed in Escherichia coli to produce a recombinant fusion protein with glutathione S-transferase (GST). The purified recombinant protein was recognized by sera of patients with proven paracoccidioidomycosis and not by sera of healthy individuals. The recombinant 45-kDa protein was shown to be catalytically active; FMD activity was detected in P. brasiliensis yeast and mycelium.     

Human and Escherichia coli cyclophilins: sensitivity to inhibition by the immunosuppressant cyclosporin A correlates with a specific tryptophan residue

The human T-cell protein cyclophilin shows high affinity for and is the proposed target of the major immunosuppressant drug cyclosporin A (CsA). Cyclophilin also has peptidyl prolyl cis-trans isomerase activity that is inhibited by CsA with an IC50 of 6 nM, while by contrast a homologous PPIase from Escherichia coli has been found to be much less sensitive to CsA, shown here to be 500-fold less potent at an IC50 of 3000 nM. This E. coli rotamase lacks the single highly conserved tryptophan residue of eukaryotic cyclophilins, and we show here that mutation of the natural F112 to W112 enhances E. coli rotamase susceptibility to CsA inhibition by 23-fold. Correspondingly, the human W121 mutations to F121 or A121 yield cyclophilins with 75- and 200-fold decreased sensitivity to CsA, while kcat/Km values of rotamase activity in a tetrapeptide assay drop only 2- and 13-fold, respectively. This complementary gain and loss of CsA sensitivity to mutation to or from tryptophan validate the indole side chain as a major determinant in immunosuppressant drug recognition and the separation of PPIase catalytic efficiency from CsA affinity.     

Expression and purification of the recombinant SALT lectin from rice (Oryza sativa L.)

The SALT protein is a 14.5 kDa mannose-binding lectin, originally described as preferentially expressed in rice plant roots in response to NaCl stress. Recombinant SALT lectin was produced in Escherichia coli from a cDNA clone encoding protein. After isopropyl-beta-d-thiogalactopyranoside induction, the expression level achieved was 23% of the soluble protein. The recombinant agglutinin was purified by a single-step process by dialyses against a high concentrated salt solution. After purification, hemagglutination assays of rabbit erythrocytes revealed that the recombinant SALT protein is a potent agglutinin (0.078 microg ml(-1) minimal concentration). The purified recombinant lectin was also used for comparative estimation of native protein amounts in protein extracts from rice plants by Western blot assay.     

Prokaryotic expression of chicken infectious anemia apoptin protein and characterization of its polyclonal antibodies

In the present study recombinant VP3 (rVP3) was expressed in E. coli BL21 (DE3) (pLysS) and its polyclonal antibodies were characterized. SDS-PAGE analysis revealed that the expression of recombinant protein was maximum when induced with 1.5 mM IPTG for 6 h at 37 degrees C. The 6xHis-tagged fusion protein was purified on Ni-NTA and confirmed by Western blot using CAV specific antiserum. Rabbits were immunized with purified rVP3 to raise anti-VP3 polyclonal antibodies. Polyclonal serum was tested for specificity and used for confirming expression of VP3 in HeLa cells transfected with pcDNA.cav.vp3 by indirect fluorescent antibody test (IFAT), flow cytometry and Western blot. Available purified rVP3 and polyclonal antibodies against VP3 may be useful to understand its functions which may lead to application of VP3 in cancer therapeutics.     

Expression and partial purification of human prolactin in Escherichia coli

1. Human prolactin has been expressed in Escherichia coli. A cDNA fragment coding for the signal sequence and the full length prolactin molecule was cloned into the expression vector pUR291 which directs the synthesis of a beta-galactosidase prolactin fusion protein when expressed in E. coli. 2. Cultures of E. coli harbouring the recombinant plasmid pJMBG62 produced a fusion protein of the appropriate molecular weight which was detected by Western blot analysis using a polyclonal antibody raised against pituitary-derived human prolactin. 3. The fusion protein was isolated from inclusion bodies in a partially pure form and it was used as immunogen to raise antibodies against human prolactin. 4. When this partially purified fusion protein was injected into rabbits it generated antisera with good prolactin titres in animals which were rested for one year following a disappointing primary immunization with purified human prolactin.     

Cloning,expression and characterization of attachment‐invasion locus protein (Ail) of Yersinia enterocolitica and its utilization in rapid detection by immunoassays

Aims: Rapid detection of pathogenic Yersinia enterocolitica isolates by using antisera raised against recombinant attachment‐invasion locus (Ail) protein. Methods and Results: The complete gene (471 bp) encoding for the Ail protein was amplified by PCR and cloned in pQE 30 UA vector. The recombinant clones were selected by polymerase chain reaction (PCR). Recombinant protein was expressed using induction with 1 mmol l^?1 final concentration of isopropylthiogalactoside (IPTG). Polyclonal antibodies were raised in mice against this purified recombinant protein. An indirect plate ELISA was standardized based on rAil protein for the detection of Y. enterocolitica. Western blot analysis with the sera raised against recombinant Ail protein exhibited reaction at 17 kDa region of the native Ail protein present in pathogenic Y. enterocolitica standard strains and strains isolated from pork samples suggesting that the antigenicity of recombinant Ail protein was similar to that of native Ail protein. Nonpathogenic Y. enterocolitica and the other species of Yersinia, namely, Y. pseudotuberculosis, Y. intermedia, Y. kristenseni, Y. fredrickseni and also the Enterobacteriaceae organisms tested were not found reacting to polyclonal antisera against this recombinant Ail protein. Conclusion: The antibodies raised against recombinant Ail protein could specifically identify pathogenic Y. enterocolitica strains both by indirect plate ELISA and Western blot immunoassay. Significance and Impact of the Study: The method developed in this study may find application in the detection of pathogenic Y. enterocolitica not only from food and environmental samples but also from clinical samples.