heparin-Sepharose Chromatography Research Articles

Expression of Untagged Arrestins in E. coli and Their Purification.

Purified arrestin proteins are necessary for biochemical, biophysical, and structural studies of these versatile regulators of cell signaling. Described herein is a basic protocol for arrestin expression in Escherichia coli and purification of tag-free wild-type and mutant arrestins. The method includes ammonium sulfate precipitation of arrestins from cell lysates, followed by Heparin-Sepharose chromatography. Depending on the arrestin type and/or mutations, the next step is Q-Sepharose or SP-Sepharose chromatography. In many cases, the nonbinding column is used as a filter to bind contaminants without retaining arrestin. In some cases, both chromatographic steps must be performed sequentially to achieve high purity. Purified arrestins can be concentrated up to 10 mg/ml, remain fully functional, and withstand several cycles of freezing and thawing, provided that the overall salt concentration is maintained at or above physiological levels. © 2023 Wiley Periodicals LLC. Basic Protocol: Large-scale expression and purification of arrestins Alternate Protocol: Purification of arrestin-3 and truncated form of arrestin-1-(1-378) Support Protocol: Small-scale test expression of wild-type and mutant arrestins in E. coli.

Feature of Adhesins Produced by Human Clinical Isolates of Mycobacterium intracellulare, Mycobacterium intracellulare subsp. chimaera and Closely Related Species.

The Mycobacterium avium complex includes two closely related species, Mycobacterium avium and Mycobacterium intracellulare. They are opportunistic pathogens in humans and responsible for severe disease in a wide variety of animals. Yet, little is known about factors involved in their pathogenicity. Here, we identified, purified and characterized adhesins belonging to the heparin-binding hemagglutinin (HBHA) and laminin-binding protein (LBP) family from M. intracellulare ATCC13950 and examined clinical isolates from patients with different pathologies associated with M. intracellulare infection for the presence and conservation of HBHA and LBP. Using a recombinant derivative strain of M. intracellulare ATCC13950 producing green fluorescent protein and luciferase, we found that the addition of heparin inhibited mycobacterial adherence to A549 cells, whereas the addition of laminin enhanced adherence. Both HBHA and LBP were purified by heparin-Sepharose chromatography and their methylation profiles were determined by mass spectrometry. Patients with M. intracellulare infection mounted strong antibody responses to both proteins. By using PCR and immunoblot analyses, we found that both proteins were highly conserved among all 17 examined clinical M. intracellulare isolates from patients with diverse disease manifestations, suggesting a conserved role of these adhesins in M. intracellulare virulence in humans and their potential use as a diagnostic tool.

Migration of chicken egg-white protein ovalbumin-related protein X and its alteration in heparin-binding affinity during embryogenesis of fertilized egg

Ovalbumin-related protein X (OVAX) is a 50kDa egg-white protein which has heparin-binding affinity. In this study, migration of OVAX and its heparin-binding performance during embryogenesis of fertilized egg were investigated to explore a possible involvement in chick embryo development. Western blotting of egg yolks at different stages using an anti-OVAX antibody showed that OVAX accumulates in yolk during incubation of fertilized egg. Immunohistochemical analysis of embryo resided in 10-day-incubated eggs showed that OVAX existed in almost all tissues of the embryo. These suggest that OVAX is incorporated from egg white into the embryo through yolk sac. Heparin-sepharose chromatography, isothermal titrating calorimetry using fondaparinux as a ligand, and zeta potential measurement indicated that OVAX retained the heparin-binding affinity (Kd = 0.185 ± 0.037) even after 10 D incubation of fertilized egg, although the affinity was slightly decreased during egg incubation because of acidification of molecular surface charge. In conclusion, although heparin-binding ability of egg-white OVAX slightly decreases during embryogenesis, OVAX incorporated into embryo can retain heparin-binding affinity. Our findings provide a new insight that OVAX participates in the functions of heparin during embryogenesis.

JNJ-64179375 Inhibits Exosite I-Mediated Thrombin Activity While Preserving Exosite II and Active Site Function in Vitro

Anticoagulation requires a careful balance to achieve antithrombotic efficacy without disrupting normal hemostasis. Although current generation direct oral anticoagulants (DOACs) offer advantages over traditional warfarin or heparin therapy they still carry a bleeding risk, highlighting an unmet need for novel anticoagulants with improved therapeutic indices. DOACs such as apixaban and dabigatran bind to the active sites of their target proteases thereby inhibiting proteolysis of all substrates. In contrast, the recombinant antibody JNJ-64179375 (JNJ-9375) specifically binds to the exosite I region of thrombin without inhibiting active site function. Interestingly, JNJ-9375 has demonstrated an improved therapeutic index compared to apixaban in preclinical models of venous thrombosis (Chintala, M et al. Res Pract Thromb Haemost, 2017:1 (suppl 1): P201). To understand how the mechanism of action of JNJ-9375 contributes to its improved therapeutic index in preclinical models, we evaluated the impact of JNJ-9375 on the activity of thrombin toward physiological substrates in vitro. Fibrinogen is the primary in vivo substrate of thrombin and binds to it via exosite I. In a turbidimetric assay JNJ-9375 dose-dependently inhibited fibrinogen cleavage, reflecting direct competition between fibrinogen and JNJ-9375 for exosite I binding. Factor V (FV), Factor VIII (FVIII), and Factor XI (FXI) are activated by thrombin in positive feedback reactions. Exosites I and II in thrombin have been differentially implicated in its action on Factors V and VIII. Accordingly, JNJ-9375 potently inhibited thrombin-dependent FV activation while exhibiting a more modest inhibition of FVIII activation as determined by quantitative Western blotting. Activation of FXI by thrombin, another exosite I-mediated reaction, was reduced approximately 8-fold in the presence of JNJ-9375. Thrombin also interacts with the anticoagulant regulators Protein C and antithrombin III (ATIII). Thrombin activates Protein C through an exosite I-mediated interaction with thrombomodulin (TM). In chromogenic assays JNJ-9375 reduced the TM-dependent activation of Protein C approximately 7-fold. Plasma supplemented with JNJ-9375 also demonstrated a modest resistance to TM-dependent Protein C activation measured in thrombin generation assays. ATIII inactivates thrombin by binding to the active site of thrombin in a suicide substrate reaction. Despite this reaction being independent of exosite I binding the rate of thrombin inactivation by ATIII was reduced approximately 3-fold in the presence of JNJ-9375, indicating an allosteric effect of exosite I binding on the reactivity of thrombin with ATIII. In contrast to exosite I-dependent reactions, JNJ-9375 had no apparent effect on the exosite II-dependent interaction of thrombin with heparin. Pre-incubation with JNJ-9375 did not alter the elution profile of thrombin in heparin sepharose chromatography. Furthermore, both unfractionated heparin and low molecular weight heparin dramatically accelerated inactivation of thrombin by ATIII in the presence of JNJ-9375, indicating that exosite II function was preserved. In summary these in vitro studies illustrate a global mechanism wherein JNJ-9375 attenuates the activity of thrombin toward both pro- and anti-coagulant components of the coagulation cascade through competitive binding to the exosite I region. This distinct mechanism in which JNJ-9375 modulates thrombin without inhibiting its proteolytic activity or exosite II-mediated activity differentiates JNJ-9375 from active site-specific DOACs. We hypothesize that the residual catalytic activity of JNJ-9375-bound thrombin enables antithrombotic efficacy without the concomitant bleeding risk seen with current generation DOACs, resulting in an improved therapeutic index. DisclosuresKrishnaswamy:Baxalta: Consultancy; Portola: Research Funding; Janssen Research & Development: Research Funding.

SHuffle™ T7 strain is capable of producing high amount of recombinant human fibroblast growth factor-1 (rhFGF-1) with proper physicochemical and biological properties

BackgroundHuman fibroblast growth factor-1 (FGF-1) has powerful mitogenic activities in a variety of cell types and plays significant roles in many physiological processes e.g. angiogenesis and wound healing. There is increasing demand for large scale production of recombinant human FGF-1 (rhFGF-1), in order to investigate the potential medical use. In the present study, we explored SHuffle™ T7 strain for production of rhFGF-1. MethodsA synthetic gene encoding Met-140 amino acid form of human FGF-1 was utilized for expression of the protein in three different E. coli hosts (BL21 (DE3), Rosetta-gami™ 2(DE3), SHuffle™ T7). Total expressions and soluble/insoluble expression ratios of rhFGF-1 in different hosts were analyzed and compared. Soluble rhFGF-1 produced in SHuffle™ T7 cells was purified using one-step heparin-Sepharose affinity chromatography and characterized by a variety of methods for physicochemical and biological properties. ResultsThe highest level of rhFGF-1 expression and maximum soluble/insoluble ratio were achieved in SHuffle™ T7 strain. Using a single-step heparin-Sepharose chromatography, about 1500mg of purified rhFGF-1 was obtained from one liter of the culture, representing purification yield of ∼70%. The purified protein was reactive toward anti-FGF-1 ployclonal antibody in immunoblotting. Mass spectrometry confirmed the protein had expected amino acid sequence and molecular weight. In reverse-phase high-performance liquid chromatography (RP-HPLC), the protein displayed the same retention time with the human FGF-1 standard, and purity of 94%. Less than 0.3% of the purified protein was comprised of oligomers and/or aggregates as judged by high-performance size-exclusion chromatography (HP-SEC). Secondary and tertiary structures of the protein, investigated by circular dichroism and intrinsic fluorescence spectroscopy methods, respectively, represented native folding of the protein. The purified rhFGF-1 was bioactive and stimulated proliferation of NIH 3T3 cells with EC50 of 0.84ng/mL. ConclusionAlthough SHuffle™ T7 has been introduced for production of disulfide-bonded proteins in cytoplasm, we herein successfully recruited it for high yield production of soluble and bioactive rhFGF-1, a protein with 3 free cysteine and no disulfide bond. To our knowledge, this is the highest-level of rhFGF-1 expression in E. coli reported so far. Extensive physicochemical and biological analysis showed the protein had similar characteristic to authentic FGF-1.

Convenient expression, purification and quantitative liquid chromatography-tandem mass spectrometry-based analysis of TET2 5-methylcytosine demethylase

5-Methylcytosine within CpG islands in DNA plays a crucial role in epigenetic transcriptional regulation during metazoan development. Recently, it has been established that the Ten-Eleven Translocation (TET) family, Fe(II)- and 2-oxoglutarate (2OG/αKG)-dependent oxygenases initiate 5-methylcytosine demethylation by iterative oxidation reactions. Mutations in the TET2 gene are frequently detected in patients with myeloid malignancies. Here, we describe the cloning of untagged human TET2 demethylase using Gateway technology and its efficient expression in E. coli. The untagged TET2 enzyme was purified using cation exchange and heparin sepharose chromatography. In addition, a reliable quantitative liquid chromatography-tandem mass spectrometry-based assay was utilized to analyze the activity of TET2 oxygenase. This assay was further used to analyze the activity of a number of clinical TET2 variants with mutations in the 2OG binding sites. Our results demonstrate that the activity of one TET2 mutant, TET2-R1896S, can be restored using an excess of 2OG in the reaction mixture. These studies suggest that dietary 2OG supplements, which are commonly used for several other conditions, may be used to treat some patients with myeloid malignancies harboring TET2-R1896S mutation. Results described in this paper serve as a foundation for better characterization of wild type as well as mutant TET2 demethylases.

Examination of Glycosaminoglycan Binding Sites on the XCL1 Dimer.

Known for its distinct metamorphic behavior, XCL1 interconverts between a canonical chemokine folded monomer (XCL1mon) that interacts with the receptor, XCR1, and a unique dimer (XCL1dim) that interacts with glycosaminoglycans and inhibits HIV-1 activity. This study presents the first detailed analysis of the GAG binding properties of XCL1dim. Basic residues within a conformationally selective dimeric variant of XCL1 (W55D) were mutated and analyzed for their effects on heparin binding. Mutation of Arg23 and Arg43 greatly diminished the level of heparin binding in both heparin Sepharose chromatography and surface plasmon resonance assays. To assess the contributions of different GAG structures to XCL1 binding, we developed a solution fluorescence polarization assay and correlated affinity with the length and level of sulfation of heparan sulfate oligosaccharides. It was recently demonstrated that the XCL1 GAG binding form, XCL1dim, is responsible for preventing HIV-1 infection through interactions with gp120. This study defines a GAG binding surface on XCL1dim that includes residues that are important for HIV-1 inhibition.

Arrestin expression in E. coli and purification.

Purified arrestin proteins are necessary for biochemical, biophysical, and crystallographic studies of these versatile regulators of cell signaling. Described herein is a basic protocol for arrestin expression in E. coli and purification of the tag-free wild-type and mutant arrestins. The method includes ammonium sulfate precipitation of arrestins from cell lysates, followed by heparin-Sepharose chromatography. Depending on the arrestin type and/or mutations, the next step is Q-Sepharose or SP-Sepharose chromatography. In many cases the nonbinding column is used as a filter to bind contaminants without retaining arrestin. In some cases both chromatographic steps must be performed sequentially to achieve high purity. Purified arrestins can be concentrated up to 10 mg/ml, remain fully functional, and withstand several cycles of freezing and thawing, provided that overall salt concentration is maintained at or above physiological levels.

A purification strategy for analysis of the DNA/RNA-associated sub-proteome from chloroplasts of mustard cotyledons.

Plant cotyledons are a tissue that is particularly active in plastid gene expression in order to develop functional chloroplasts from pro-plastids, the plastid precursor stage in plant embryos. Cotyledons, therefore, represent a material being ideal for the study of composition, function and regulation of protein complexes involved in plastid gene expression. Here, we present a pilot study that uses heparin-Sepharose and phospho-cellulose chromatography in combination with isoelectric focussing and denaturing SDS gel electrophoresis (two-dimensional gel electrophoresis) for investigating the nucleic acids binding sub-proteome of mustard chloroplasts purified from cotyledons. We describe the technical requirements for a highly resolved biochemical purification of several hundreds of protein spots obtained from such samples. Subsequent mass spectrometry of peptides isolated out of cut spots that had been treated with trypsin identified 58 different proteins within 180 distinct spots. Our analyses indicate a high enrichment of proteins involved in transcription and translation and, in addition, the presence of massive post-translational modification of this plastid protein sub-fraction. The study provides an extended catalog of plastid proteins from mustard being involved in gene expression and its regulation and describes a suitable purification strategy for further analysis of low abundant gene expression related proteins.

Identification and characterization of chloroplast casein kinase II from Oryza sativa (rice).

Plastid casein kinase II is an important regulator of transcription, posttranscriptional processes, and, most likely, different metabolic functions in dicotyledonous species. Here we report the identification and characterization of pCKII from the monocotyledonous species Oryza sativa. OspCKII activity was enriched from isolated rice chloroplasts using heparin-Sepharose chromatography, in which it co-elutes with the transcriptionally active chromosome (TAC) and several ribosomal proteins. Inclusion mass scanning of the kinase-active fraction identified the gene model for OspCKII. Transient expression of GFP fused to the 184 N-terminal amino acids of the OspCKII sequence in rice confirmed the chloroplastic localization of the kinase. OspCKII activity shows the characteristic features of casein kinase II, such as the utilization of GTP as phosphate donor, inhibition by low concentrations of heparin and poly-lysine, and utilization of the canonical pCKII motif E-S-E-G-E in the model substrate RNP29. Phosphoproteome analysis of a protein extract from rice leaves combined with a meta-analysis with published phosphoproteomics data revealed differences in the target protein spectrum between rice and Arabidopsis. Consistently, several pCKII phosphorylation sites in dicotyledonous plants are not conserved in monocots and algae, suggesting that details of pCKII regulation in plastids have changed during evolution.

The peptide microarray "ChloroPhos1.0" identifies new phosphorylation targets of plastid casein kinase II (pCKII) in Arabidopsis thaliana.

We report the development of a peptide microarray based on previously determined phosphorylation sites in chloroplast proteins. Altogether, 905 peptides were spotted as 15mers in nine replicates onto glass slides. We used the microarray for in vitro phosphorylation experiments and specifically assessed the peptide substrate spectrum of chloroplast casein kinase II (pCKII). To this end, native pCKII from Arabidopsis thaliana and Sinapis alba chloroplasts was enriched by Heparin-Sepharose chromatography and its activity on the microarray was compared to the activity of a recombinant Arabidopsis pCKII. All three kinase preparations phosphorylated a similar set of peptides that were clearly distinct from those phosphorylated by bovine heart protein kinase A (PKA) in control experiments. The majority of the pCKII phosphorylation targets are involved in plastid gene expression, supporting the earlier denomination of pCKII as plastid transcription kinase (PTK). In addition we identified Alb3 as pCKII substrate that is essential for the integration of light-harvesting complex subunits (LHC) into the thylakoid membrane. Plastid CKII phosphorylation activity was characterized in greater detail in vitro with recombinant wildtype Alb3 and phosphorylation site mutants as substrates, establishing S424 as the pCKII phosphorylation site. Our data show that the peptide microarray ChloroPhos1.0 is a suitable tool for the identification of new kinase downstream targets in vitro that can be validated subsequently by in vivo experiments.

Identification of a small molecule that stabilizes lipoprotein lipase in vitro and lowers triglycerides in vivo

Patients at increased cardiovascular risk commonly display high levels of plasma triglycerides (TGs), elevated LDL cholesterol, small dense LDL particles and low levels of HDL-cholesterol. Many remain at high risk even after successful statin therapy, presumably because TG levels remain high. Lipoprotein lipase (LPL) maintains TG homeostasis in blood by hydrolysis of TG-rich lipoproteins. Efficient clearance of TGs is accompanied by increased levels of HDL-cholesterol and decreased levels of small dense LDL. Given the central role of LPL in lipid metabolism we sought to find small molecules that could increase LPL activity and serve as starting points for drug development efforts against cardiovascular disease. Using a small molecule screening approach we have identified small molecules that can protect LPL from inactivation by the controller protein angiopoietin-like protein 4 during incubations in vitro. One of the selected compounds, 50F10, was directly shown to preserve the active homodimer structure of LPL, as demonstrated by heparin-Sepharose chromatography. On injection to hypertriglyceridemic apolipoprotein A-V deficient mice the compound ameliorated the postprandial response after an olive oil gavage. This is a potential lead compound for the development of drugs that could reduce the residual risk associated with elevated plasma TGs in dyslipidemia.

Novel Feature of Mycobacterium avium subsp. paratuberculosis, Highlighted by Characterization of the Heparin-Binding Hemagglutinin Adhesin

Mycobacterium avium subsp. paratuberculosis comprises two genotypically defined groups, known as the cattle (C) and sheep (S) groups. Recent studies have reported phenotypic differences between M. avium subsp. paratuberculosis groups C and S, including growth rates, infectivity for macrophages, and iron metabolism. In this study, we investigated the genotypes and biological properties of the virulence factor heparin-binding hemagglutinin adhesin (HBHA) for both groups. In Mycobacterium tuberculosis, HBHA is a major adhesin involved in mycobacterium-host interactions and extrapulmonary dissemination of infection. To investigate HBHA in M. avium subsp. paratuberculosis, we studied hbhA polymorphisms by fragment analysis using the GeneMapper technology across a large collection of isolates genotyped by mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) and IS900 restriction fragment length polymorphism (RFLP-IS900) analyses. Furthermore, we analyzed the structure-function relationships of recombinant HBHA proteins of types C and S by heparin-Sepharose chromatography and surface plasmon resonance (SPR) analyses. In silico analysis revealed two forms of HBHA, corresponding to the prototype genomes for the C and S types of M. avium subsp. paratuberculosis. This observation was confirmed using GeneMapper on 85 M. avium subsp. paratuberculosis strains, including 67 strains of type C and 18 strains of type S. We found that HBHAs from all type C strains contain a short C-terminal domain, while those of type S present a long C-terminal domain, similar to that produced by Mycobacterium avium subsp. avium. The purification of recombinant HBHA from M. avium subsp. paratuberculosis of both types by heparin-Sepharose chromatography highlighted a correlation between their affinities for heparin and the lengths of their C-terminal domains, which was confirmed by SPR analysis. Thus, types C and S of M. avium subsp. paratuberculosis may be distinguished by the types of HBHA they produce, which differ in size and adherence properties, thereby providing new evidence that strengthens the genotypic differences between the C and S types of M. avium subsp. paratuberculosis.

Purification of native HBHA from Mycobacterium avium subsp. paratuberculosis

BackgroundParatuberculosis remains today a major global problem in animal health, especially for dairy cattle. However, the diagnosis of its etiologic agent, Mycobacterium avium subsp. paratuberculosis (Map), still lacks sensitivity because of the lack of available antigens. Little is known about the virulence factors for this pathogen. In this study we have developed a method to produce and purify the heparin-binding hemagglutinin (HBHA), a major adhesin of Mycobacteria, from a culture of Map.FindingsFor this extremely slow-growing Mycobacterium, a culture was established in a 3-liter bioreactor. Using the bioreactor the amount of the Map biomass was increased 5-fold compared to a classical culture in flasks. The map-HBHA was purified from a Map lysate by heparin-Sepharose chromatography on HiTrap columns. Binding of map-HBHA onto heparin-Sepharose can be reduced in the presence of salt. Consequently, all steps of sample preparation and column equilibration were carried out in 20 mM Tris–HCl (pH 7.2). The map-HBHA was eluted by a linear NaCl gradient. High resolution mass spectrometry analyses revealed that the native form of map-HBHA has posttranslational modifications, including the removal of the initiation methionine, acetylation of the alanine residue at the N-terminal extremity and the presence of methylated lysines in the C-terminal domain of the protein.ConclusionsAn optimized culture of Map in a bioreactor was established to purify the native map-HBHA from a Map lysate by heparin-Sepharose chromatography. The availability of this antigen offers the possibility to study the structure of the protein and to examine its role in pathogenicity, in particular to better understand the specific interactions of Map with the intestinal tissue. The map-HBHA obtained in its native immunogenic form may also be useful to improve the diagnostic test, especially for the development of a new T-cell-based interferon gamma release assays.

Chaperonin TRiC assists the refolding of sperm-specific glyceraldehyde-3-phosphate dehydrogenase

The cytosolic chaperonin TRiC was isolated from ovine testes using ultracentrifugation and heparin-Sepharose chromatography. The molecular mass of the obtained preparation was shown to exceed 900 kDa (by Blue Native PAGE). SDS–PAGE yielded a set of bands in the range of 50–60 kDa. Electron microscopy examination revealed ring-shaped complexes with the outer diameter of 15 nm and the inner diameter of approximately 6 nm. The results suggest that the purified chaperonin is an oligomeric complex composed of two 8-membered rings. The chaperonin TRiC was shown to assist an ATP-dependent refolding of recombinant forms of sperm-specific glyceraldehyde-3-phosphate dehydrogenase, an enzyme that is expressed only in precursor cells of the sperms in the seminiferous tubules of the testes. In contrast, TRiC did not influence the refolding of muscle isoform of glyceraldehyde-3-phosphate dehydrogenase and assisted the refolding of muscle lactate dehydrogenase by an ATP-independent mechanism. The obtained results suggest that TRiC is likely to be involved in the refolding of sperm-specific proteins.

Purification and characterization of SepII a new restriction endonuclease from Staphylococcus epidermidis

A Type II restriction enzyme SepII has been purified to apparent homogeneity from the gram-positive coccus, Staphylococcus epidermidis. The purification included an ammonium sulfate precipitation followed by Q-sepharose, heparin-sepharose and MonoQ column chromatography on an FPLC system. SDS-PAGE analysis showed a denatured molecular weight of 29 kDa. The effects of temperature, pH, NaCl, Mn 2+, Ca 2+, and Mg 2+ ion concentrations were studied to determine the optimal reaction conditions. The enzyme exhibits near maximal levels of activity between pH 8–10, at 10–20 mM MgCl 2, 100–150 mM NaCl and 1 mM DTT. The results also show that in NEB Buffer 3 the enzyme is active over a broad temperature range from 0 to 70 °C, and in the absence of DNA, enzyme thermostability is observed up to 50 °C for 20 min, while most of the original activity is conserved in 50% glycerol for weeks at room temperature. Single and double digestion in presence of commercial restriction enzymes of known DNA substrates (lambda, pBR322, pET21, pTrcHisB, pPB67) showed that the purified SepII recognized and cleaved the same site as EcoRV. Genomic DNA modification status was also determined.

Characterization of the Mycobacterium avium subsp. paratuberculosis laminin-binding/histone-like protein (Lbp/Hlp) which reacts with sera from patients with Crohn’s disease

Mycobacterium avium subsp. paratuberculosis ( Map) causes a chronic enteric disease in ruminants, called paratuberculosis or Johne’s disease. The current model proposes that after ingestion by the host, Map crosses the intestinal barrier via internalization by the M cells. Experimental observations suggest, however, that Map may also transcytose the intestinal wall via the enterocytes, but the mechanisms involved in this process remain poorly understood. Cytoadherence assays performed on epithelial cells with Map revealed that the addition of laminin to the cell culture increases adhesion. A Map protein was isolated by heparin–Sepharose chromatography and identified as a laminin-binding protein like. The gene encoding this protein named Lbp/Hlp was identified in the Map genome sequence at locus MAP3024 (annotated Hup B). The deduced Map Lbp/Hlp amino acid sequence reveals 80% identity with that reported for other mycobacteria. The C-terminal domain involved in adhesion is mainly composed of arginine and lysine residues modified by methylation. In vitro tests demonstrated that recombinant Lbp/Hlp binds laminin, heparin, collagen and epithelial cells. Interestingly, we found that this adhesin corresponds to the antigen described as the target of pANCA and serum antibodies of patients with Crohn’s disease.

Analysis of oligomeric protein complexes in the chloroplast sub‐proteome of nucleic acid‐binding proteins from mustard reveals potential redox regulators of plastid gene expression

Photosynthetic light quality acclimation in plants involves redox-controlled changes in plastid gene expression. To study proteins potentially involved in this regulation, we isolated low-abundant chloroplast nucleic acid-binding proteins from the crucifere mustard (Sinapis alba) and investigated if photosynthetic redox signals affect their composition and/or oligomeric structure. We purified chloroplasts from plants subjected to light quality shifts and applied organelle lysates to heparin-Sepharose chromatography followed by 2-D blue native PAGE. We studied accumulation and structure of oligomeric protein complexes and applied MS/MS to identify them. We found ten oligomeric protein complexes of higher order and eleven smaller protein complexes or spots including plastid-encoded RNA polymerase (PEP), plastid transcriptionally active chromosome proteins, RNA-binding proteins, ribosomal subunits and chaperones. A translation elongation factor was found to be the only protein displaying major differences in its amounts in response to the growth lights. Furthermore, we found a novel thioredoxin as a subunit of the PEP, a 2-Cys-peroxiredoxin complex and a (soluble) ferredoxin:NADP-oxido-reductase, which represent potential redox regulator of plastid gene expression. A T-DNA knock-out line of the thioredoxin from Arabidopsis exhibits a yellowish-pale phenotype, demonstrating that this novel PEP subunit is essential for proper plastid development.

Gentisic Acid, a Compound Associated with Plant Defense and a Metabolite of Aspirin, Heads a New Class of in Vivo Fibroblast Growth Factor Inhibitors

Fibroblast growth factors are key proteins in many intercellular signaling networks. They normally remain attached to the extracellular matrix, which confers on them a considerable stability. The unrestrained accumulation of fibroblast growth factors in the extracellular milieu, either due to uncontrolled synthesis or enzymatic release, contributes to the pathology of many diseases. Consequently, the neutralization of improperly mobilized fibroblast growth factors is of clear therapeutic interest. In pursuing described rules to identify potential inhibitors of these proteins, gentisic acid, a plant pest-controlling compound, an aspirin and vegetarian diet common catabolite, and a component of many traditional liquors and herbal remedies, was singled out as a powerful inhibitor of fibroblast growth factors. Gentisic acid was used as a lead to identify additional compounds with better inhibitory characteristics generating a new chemical class of fibroblast growth factor inhibitors that includes the agent responsible for alkaptonuria. Through low and high resolution approaches, using representative members of the fibroblast growth factor family and their cell receptors, it was shown that this class of inhibitors may employ two different mechanisms to interfere with the assembly of the signaling complexes that trigger fibroblast growth factor-driven mitogenesis. In addition, we obtained evidence from in vivo disease models that this group of inhibitors may be of interest to treat cancer and angiogenesis-dependent diseases.

Expression and characterization of Kunitz domain 3 and C-terminal of human tissue factor pathway inhibitor-2

Human tissue factor pathway inhibitor-2 (hTFPI-2) is a serine protease inhibitor and its inhibitory activity is enhanced by heparin. The Kunitz domain 3 and Cterminal of hTFPI-2 (hTFPI-2/KD3C), which has the activity toward heparin calcium, have been successfully expressed in Pichia pastoris and purified by SPSepharose and heparin-Sepharose chromatography. The Fourier transformed infrared spectroscopy (FTIR), Raman spectroscopy, and circular dichroism (CD) experiment results implied that hTFPI-2/KD3C contained small contents of alpha-helix and beta-strand, but large amounts of random coil and two kinds of disulfide bonds, gauche-gauche-gauche (ggg) and trans-gauchetrans (tgt). The interaction of hTFPI-2/KD3C with heparin calcium was investigated by CD. It was found that heparin calcium induced b-strands in hTFPI-2/ KD3C to different extents depending on the ratio of hTFPI-2/KD3C and heparin calcium.