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N-terminal Sequence (n-terminal + sequence)
Selected AbstractsDe novo synthesis, uptake and proteolytic processing of lipocalin-type prostaglandin D synthase, ,-trace, in the kidneysFEBS JOURNAL, Issue 23 2009Nanae Nagata Lipocalin-type prostaglandin D synthase (L-PGDS) is a multifunctional protein that produces prostaglandin D2 and binds and transports various lipophilic substances after secretion into various body fluids as ,-trace. L-PGDS has been proposed to be a useful diagnostic marker for renal injury associated with diabetes or hypertension, because the urinary and plasma concentrations are increased in patients with these diseases. However, it remains unclear whether urinary L-PGDS is synthesized de novo in the kidney or taken up from the blood circulation. In crude extracts of monkey kidney and human urine, we found L-PGDS with its original N-terminal sequence starting from Ala23 after the signal sequence, and also its N-terminal-truncated products starting from Gln31 and Phe34. In situ hybridization and immunohistochemical staining with monoclonal antibody 5C11, which recognized the amino-terminal Ala23,Val28 loop of L-PGDS, revealed that both the mRNA and the intact form of L-PGDS were localized in the cells of Henle's loop and the glomeruli of the kidney, indicating that L-PGDS is synthesized de novo in these tissues. However, truncated forms of L-PGDS were found in the lysosomes of tubular cells, as visualized by immunostaining with 10A5, another monoclonal antibody, which recognized the three-turn ,-helix between Arg156 and Thr173. These results suggest that L-PGDS is taken up by tubular cells and actively degraded within their lysosomes to produce the N-terminal-truncated form. Structured digital abstract ,,MINT-7266187: L-PGDS (uniprotkb:P41222) and Cathepsin D (uniprotkb:Q4R4P0) colocalize (MI:0403) by fluorescence microscopy (MI:0416) ,,MINT-7266176: L-PGDS (uniprotkb:P41222) and Cathepsin B (uniprotkb:Q4R5M2) colocalize (MI:0403) by fluorescence microscopy (MI:0416) [source] Secondary structure assignment of mouse SOCS3 by NMR defines the domain boundaries and identifies an unstructured insertion in the SH2 domainFEBS JOURNAL, Issue 23 2005Jeffrey J. Babon SOCS3 is a negative regulator of cytokine signalling that inhibits Janus kinase-signal transduction and activator of transcription (JAK-STAT) mediated signal tranduction by binding to phosphorylated tyrosine residues on intracellular subunits of various cytokine receptors, as well as possibly the JAK proteins. SOCS3 consists of a short N-terminal sequence followed by a kinase inhibitory region, an extended SH2 domain and a C-terminal suppressor of cytokine signalling (SOCS) box. SOCS3 and the related protein, cytokine-inducible SH2-containing protein, are unique among the SOCS family of proteins in containing a region of mostly low complexity sequence, between the SH2 domain and the C-terminal SOCS box. Using NMR, we assigned and determined the secondary structure of a murine SOCS3 construct. The SH2 domain, unusually, consists of 140 residues, including an unstructured insertion of 35 residues. This insertion fits the criteria for a PEST sequence and is not required for phosphotyrosine binding, as shown by isothermal titration calorimetry. Instead, we propose that the PEST sequence has a functional role unrelated to phosphotyrosine binding, possibly mediating efficient proteolytic degradation of the protein. The latter half of the kinase inhibitory region and the entire extended SH2 subdomain form a single ,-helix. The mapping of the true SH2 domain, and the location of its C terminus more than 50 residues further downstream than predicted by sequence homology, explains a number of previously unexpected results that have shown the importance of residues close to the SOCS box for phosphotyrosine binding. [source] Biochemical and molecular characterization of a laccase from the edible straw mushroom, Volvariella volvaceaFEBS JOURNAL, Issue 2 2004Shicheng Chen We have isolated a laccase (lac1) from culture fluid of Volvariella volvacea, grown in a defined medium containing 150 µm CuSO4, by ion-exchange and gel filtration chromatography. Lac1 has a molecular mass of 58 kDa as determined by SDS/PAGE and an isoelectric point of 3.7. Degenerate primers based on the N-terminal sequence of purified lac1 and a conserved copper-binding domain were used to generate cDNA fragments encoding a portion of the lac1 protein and RACE was used to obtain full-length cDNA clones. The cDNA of lac1 contained an ORF of 1557 bp encoding 519 amino acids. The amino acid sequence from Ala25 to Asp41 corresponded to the N-terminal sequence of the purified protein. The first 24 amino acids are presumed to be a signal peptide. The expression of lac1 is regulated at the transcription level by copper and various aromatic compounds. RT-PCR analysis of gene transcription in fungal mycelia grown on rice-straw revealed that, apart from during the early stages of substrate colonization, lac1 was expressed at every stage of the mushroom developmental cycle defined in this study, although the levels of transcription varied considerably depending upon the developmental phase. Transcription of lac1 increased sharply during the latter phase of substrate colonization and reached maximum levels during the very early stages (primordium formation, pinhead stage) of fruit body morphogenesis. Gene expression then declined to ,,20,30% of peak levels throughout the subsequent stages of sporophore development. [source] The archaeal flagellum: a different kind of prokaryotic motility structureFEMS MICROBIOLOGY REVIEWS, Issue 2 2001Nikhil A Thomas Abstract The archaeal flagellum is a unique motility apparatus distinct in composition and likely in assembly from the bacterial flagellum. Gene families comprised of multiple flagellin genes co-transcribed with a number of conserved, archaeal-specific accessory genes have been identified in several archaea. However, no homologues of any bacterial genes involved in flagella structure have yet been identified in any archaeon, including those archaea in which the complete genome sequence has been published. Archaeal flagellins possess a highly conserved hydrophobic N-terminal sequence that is similar to that of type IV pilins and clearly unlike that of bacterial flagellins. Also unlike bacterial flagellins but similar to type IV pilins, archaeal flagellins are initially synthesized with a short leader peptide that is cleaved by a membrane-located peptidase. With recent advances in genetic transfer systems in archaea, knockouts have been reported in several genes involved in flagellation in different archaea. In addition, techniques to isolate flagella with attached hook and anchoring structures have been developed. Analysis of these preparations is under way to identify minor structural components of archaeal flagella. This and the continued isolation and characterization of flagella mutants should lead to significant advances in our knowledge of the composition and assembly of archaeal flagella. [source] The Rubino test for leprosy is a ,2 -glycoprotein 1-dependent antiphospholipid reactionIMMUNOLOGY, Issue 1 2000A. Panunto-Castelo Summary We describe the isolation and identification of three components required for the Rubino reaction (RR), which is the rapid sedimentation of formalinized sheep red-blood cells (SRBC) initiated by serum from leprosy patients with defective Mycobacterium leprae -specific cell immunity. The Rubino reaction factor (RRF) required for this phenomenon, previously identified as an immunoglobulin M (IgM), was purified from leprosy patient serum by adsorption to formalinized SRBC. Purified RRF IgM, when added to formalinized SRBC, did not produce a positive RR. However, when the contact was carried out in the presence of normal human serum (NHS), cells rapidly sedimented. The purified cofactor from NHS contained two components of 70 000 and 50 000 molecular weight (MW), as determined by sodium dodecyl sulphate,polyacrylamide gel electrophoresis (SDS,PAGE). The latter was recognized by the RRF IgM on immunoblot and its N-terminal sequence indicated that it was ,2 -glycoprotein 1 (,2 -GP1), an anionic phospholipid-binding protein. Methanol-treated formalinized SRBC did not support the RR. Thin-layer chromatography of an extract of membranes indicated that the SRBC ligand was a cell-surface phospholipid. Cardiolipin inhibited the RR. These data demonstrate that the RR involves a trimolecular interaction in which IgM, ,2 -GP1 and an SRBC phospholipid participate. By analogy with the antiphospholipid antibodies (anti-PL) that occur in autoimmune processes, serum samples from 29 systemic lupus erythematosus patients with high levels of anticardiolipin antibodies were submitted to the RR. A positive RR was obtained for 45% (13 of 29 patients). These results modify the paradigm of the absolute specificity of the RR for leprosy and demonstrate that RRF IgM is a ,2 -GP1-dependent anti-PL. [source] Determination of wheat quality by mass spectrometry and multivariate data analysisRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 21 2002David Mark Gottlieb Multivariate analysis has been applied as support to proteome analysis in order to implement an easier and faster way of data handling based on separation by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. The characterisation phase in proteome analysis by means of simple visual inspection is a demanding process and also insecure because subjectivity is the controlling element. Multivariate analysis offers, to a considerable extent, objectivity and must therefore be regarded as a neutral way to evaluate results obtained by proteome analysis. Proteome analysis of storage proteins from the wheat gluten complex based on two-dimensional electrophoresis and analysis of the N-terminal sequence has revealed a protein homologous to ,-gliadins, tentatively associated with quality and within the molecular weight range 27,35,kDa. Further examinations of gliadin data based on mass spectrometry revealed that quality among wheat varieties could be determined by means of principal component analysis. Further examinations by interval partial least squares made it possible to encircle an overall optimal molecular weight interval from 31.5 to 33.7,kDa. The use of multivariate analysis on data from mass spectrometry has thus shown to be a promising technique to minimize the number of two-dimensional gels within the field of proteome analysis. Copyright © 2002 John Wiley & Sons, Ltd. [source] Localization of a flavonoid biosynthetic polyphenol oxidase in vacuolesTHE PLANT JOURNAL, Issue 2 2006Eiichiro Ono Summary Aureusidin synthase, a polyphenol oxidase (PPO), specifically catalyzes the oxidative formation of aurones from chalcones, which are plant flavonoids, and is responsible for the yellow coloration of snapdragon (Antirrhinum majus) flowers. All known PPOs have been found to be localized in plastids, whereas flavonoid biosynthesis is thought to take place in the cytoplasm [or on the cytoplasmic surface of the endoplasmic reticulum (ER)]. However, the primary structural characteristics of aureusidin synthase and some of its molecular properties argue against localization of the enzyme in plastids and the cytoplasm. In this study, the subcellular localization of the enzyme in petal cells of the yellow snapdragon was investigated. Sucrose-density gradient and differential centrifugation analyses suggested that the enzyme (the 39-kDa mature form) is not located in plastids or on the ER. Transient assays using a green fluorescent protein (GFP) chimera fused with the putative propeptide of the PPO precursor suggested that the enzyme was localized within the vacuole lumen. We also found that the necessary information for vacuolar targeting of the PPO was encoded within the 53-residue N-terminal sequence (NTPP), but not in the C-terminal sequence of the precursor. NTPP-mediated ER-to-Golgi trafficking to vacuoles was confirmed by means of the co-expression of an NTPP-GFP chimera with a dominant negative mutant of the Arabidopsis GTPase Sar1 or with a monomeric red fluorescent protein (mRFP)-fused Golgi marker (an H+ -translocating inorganic pyrophosphatase of Arabidopsis). We identified a sequence-specific vacuolar sorting determinant in the NTPP of the precursor. We have demonstrated the biosynthesis of a flavonoid skeleton in vacuoles. The findings of this metabolic compartmentation may provide a strategy for overcoming the biochemical instability of the precursor chalcones in the cytoplasm, thus leading to the efficient accumulation of aurones in the flower. [source] Localization and targeting of the VP14 epoxy-carotenoid dioxygenase to chloroplast membranesTHE PLANT JOURNAL, Issue 5 2001Bao-Cai Tan Summary Abscisic acid (ABA) is a key regulator of seed dormancy and plant responses to environmental challenges. ABA is synthesized via an oxidative cleavage of 9- cis epoxy-carotenoids, the first committed and key regulatory step in the ABA biosynthetic pathway. Vp14 of maize encodes an epoxy-carotenoid dioxygenase that is soluble when expressed in E. coli. An important goal has been to determine how the soluble VP14 protein is targeted to epoxy-carotenoid substrates that are located in the thylakoid and envelope membranes of chloroplasts and other plastids. Using an in vitro chloroplast import assay, we have shown that VP14 is imported into chloroplasts with cleavage of a short stroma-targeting domain. The mature VP14 exists in two forms, one which is soluble in stroma and the other bound to thylakoid membranes. Analysis of a series of truncated VP14 mutants mapped the membrane targeting signal to the 160 amino acid N-terminal sequence. A putative amphipathic ,-helix within this region is essential, but not sufficient, for the membrane targeting. Either deletion of or insertion of helix breaking residues into this region abolished the membrane binding, whereas a chimeric protein carrying just the amphipathic region fused with bacterial glutathione S -transferase failed to associate with the thylakoid membrane. The membrane-bound VP14 was partially resistant to chaotropic washes such as 0.1 m Na2CO3 (pH 11.5) and 6 m urea. Unlabelled recombinant VP14 inhibited the tight binding of imported VP14, suggesting that VP14 is associated with specific components of the thylakoid membrane. [source] Phosphorylated Human Lectin Galectin-3: Analysis of Ligand Binding by Histochemical Monitoring of Normal/Malignant Squamous Epithelia and by Isothermal Titration CalorimetryANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 1 2009P. Szabo Summary The human lectin galectin-3 is a multifunctional effector with special functions in regulation of adhesion and apoptosis. Its unique trimodular organization includes the 12-residue N-terminal sequence, a substrate for protein kinase CK1-dependent phosphorylation. As a step towards elucidating its significance, we prepared phosphorylated galectin-3, labelled it and used it as a tool in histochemistry. We monitored normal and malignant squamous epithelia. Binding was suprabasal with obvious positive correlation to the degree of differentiation and negative correlation to proliferation. The staining pattern resembled that obtained with the unmodified lectin. Basal cell carcinomas were invariably negative. The epidermal positivity profile was akin to distribution of the desmosomal protein desmoglein, as also seen with keratinocytes in vitro. In all cases, binding was inhibitable by the presence of lactose, prompting further investigation of the activity of the lectin site by a sensitive biochemical method, i.e. isothermal titration calorimetry. The overall affinity and the individual enthalpic and entropic contributions were determined. No effect of phosphorylation was revealed. This strategic combination of histo- and biochemical techniques applied to an endogenous effector after its processing by a protein kinase thus enabled a detailed monitoring of the binding properties of the post-translationally modified lectin. It underscores the value of using endogenous lectins as a histochemical tool. The documented approach has merit for applications beyond lectinology. [source] Chiral interaction in Gly-capped N-terminal motif of 310 -helix and domino-type induction in helix senseBIOPOLYMERS, Issue 4 2006Naoki Ousaka Abstract Chiral interaction of helical peptide with chiral molecule, and concomitant induction in its helix sense have been demonstrated in optically inactive nonapeptide (1) possessing Gly at its N-terminus: H,Gly,(,ZPhe,Aib)4,OCH3 (1: ,ZPhe = Z-dehydrophenylalanine; Aib = ,-aminoisobutyric acid). Spectroscopic measurements [mainly nuclear magnetic resonance (NMR) and circular diochroism (CD)] as well as theoretical simulation have been carried out for that purpose. Peptide 1 in the 310 -helix tends to adopt preferentially a right-handed screw sense by chiral Boc- L -amino acid (Boc: t -butoxycarbonyl). Induction in the helix sense through the noncovalent chiral domino effect should be derived primarily from the complex supported by the three-point coordination on the N-terminal sequence. Thus the 310 -helical terminus consisting of only ,-amino acid residues enables chiral recognition of the Boc-amino acid molecule, leading to modulation of the original chain asymmetry. Dynamics in the helix-sense induction also have been discussed on the basis of a low-temperature NMR study. Furthermore, the inversion of induced helix sense has been achieved through solvent effects. © 2006 Wiley Periodicals, Inc. Biopolymers 83:337,351, 2006 This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Expression system for recombinant human growth hormone production from Bacillus subtilisBIOTECHNOLOGY PROGRESS, Issue 1 2009Tunçer H. Özdamar Abstract We demonstrate for the first time, an expression system mimicking serine alkaline protease synthesis and secretion, producing native form of human growth hormone (hGH) from Bacillus subtilis. A hybrid-gene of two DNA fragments, i.e., signal (pre- ) DNA sequence of B. licheniformis serine alkaline protease gene (subC) and cDNA encoding hGH, were cloned into pMK4 and expressed under deg -promoter in B. subtilis. Recombinant-hGH (rhGH) produced by B. subtilis carrying pMK4::pre(subC)::hGH was secreted. N-terminal sequence and mass spectrometry analyses of rhGH confirm the mature hGH sequence, and indicate that the signal peptide was properly processed by B. subtilis signal-peptidase. The highest rhGH concentration was obtained at t = 32 h as CrhGH = 70 mg L,1 with a product yield on substrate YrhGH/S = 9 g kg,1, in a glucose based defined medium. Fermentation characteristics and influence of hGH gene on the rhGH production were investigated by comparing B. subtilis carrying pMK4::pre(subC)::hGH with that of carrying merely pMK4. Excreted organic-acid concentrations were higher by B. subtilis carrying pMK4::pre(subC)::hGH, whereas excreted amino-acid concentrations were higher by B. subtilis carrying pMK4. The approach developed is expected to be applicable to the design of expression systems for heterologous protein production from Bacillus species. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Biological activities of Bv8 analoguesBRITISH JOURNAL OF PHARMACOLOGY, Issue 5 2005Lucia Negri The small protein Bv8, secreted by the skin of the frog Bombina variegata, belongs to a novel family of secreted proteins whose orthologues have been identified in snakes (MIT) and in mammals (prokineticins (PKs)). A characteristic feature of this protein family is the same N-terminal sequence, AVITGA, and the presence of 10 cysteines with identical spacing in the C-terminal domain. Two closely related G protein-coupled receptors that mediate signal transduction of Bv8/PKs have been cloned (PK-R1 and PK-R2). In mammals, the Bv8/PK protein family is involved in a number of biological activities such as ingestive behaviours, circadian rhythms, angiogenesis and pain sensitization. In an attempt to identify the structural determinants required for the pronociceptive activity of Bv8, we prepared Bv8 derivatives lacking one (des-Ala- Bv8) or two (des-Ala-Val -Bv8) residues from the N-terminus. des-Ala- Bv8 displayed a receptor affinity five times lower than that of Bv8, it was five times less potent in inducing [Ca2+]i transients and in causing p42/p44 MAPK phosphorylation in CHO-cells expressing PK-R1 and PK-R2. Moreover, dA-Bv8 was about 20 times less potent than Bv8 in inducing hyperalgesia in rats. The deletion of the first two amino acids of Bv8 abolished any biological activity both ,in vitro' and ,in vivo'; however, des-AlaVal -Bv8 is able to antagonize the Bv8-induced hyperalgesia, binding the PK-Rs on peripheral and central projections of the primary sensitive neurons. British Journal of Pharmacology (2005) 146, 625,632. doi:10.1038/sj.bjp.0706376 [source] Structural Investigation of the HIV-1 Envelope Glycoprotein gp160 Cleavage Site 3: Role of Site-Specific Mutations,CHEMBIOCHEM, Issue 12 2004Lucia Falcigno Dr. Abstract Proteolytic processing of HIV gp160 to produce gp120 and gp41 is performed by PC enzymes. This process is a prerequisite for the virus infectivity, since both gp120 and gp41 participate in the virus HIV-1 entry mechanism. The structure of the gp120/gp41 junction remains to be elucidated, and the structural features required for molecular recognition between HIV-1 gp160 and proteolytic enzymes have not been clarified. Furin is the best PC candidate for the gp160 proteolytic processing known to date. In previous studies on model peptides, we have shown the relevance of an N-terminal helix for the proper recognition of the gp160 processing site by furin. Here we analyze the effect of point mutations in peptides lacking a regular N-terminal helix. To this end, we present the structure,activity characterization of three peptide analogues of the HIV gp160 processing site that all present mutations in proline at positions P3 and/or P2,, while sharing the same N-terminal sequence, containing helix-breaking D -amino acids. Conformational analysis of the peptides was carried out in solution by NMR techniques, and furin's efficiency in cleaving them was measured. Structural findings are presented and discussed in relation to the different exhibited activity. [source] A low-molecular mass ribonuclease from the brown oyster mushroomCHEMICAL BIOLOGY & DRUG DESIGN, Issue 1 2005L. Xia Abstract:, A ribonuclease, with a molecular mass of 9 kDa and an N-terminal sequence resembling the sequence of a fragment of tRNA/rRNA cytosine-C5-methylase and a fragment of a alanyl-tRNA synthetase, was isolated from fresh fruiting bodies of the brown oyster mushroom Pleurotus ostreatus. The ribonuclease was purified using a very simple protocol that comprised ion-exchange chromatography on carboxymethyl (CM)-cellulose and affinity chromatography on Affi-gel blue gel. Subsequent gel filtration by fast protein liquid chromatography on Superdex 75 and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis revealed that the ribonuclease was purified after the first two chromatographic steps. The ribonuclease was adsorbed on CM-cellulose and Affi-gel blue gel. The ribonuclease exhibited the highest activity toward poly A, lower activity toward poly C, slight activity toward poly G, and indiscernible activity toward poly U. The enzyme was stimulated upon exposure to 1 ,m Mg2+ and 10 ,m Zn2+, but was inhibited by the following ions at 10 mm: Ca2+, Mg2+, Zn2+, Cu2+, Fe2+, Mn2+, and Fe3+. The ribonuclease required a pH of 8.0 and a temperature of 50,70 °C to express maximal activity. It had a Km of 60 ,m toward yeast tRNA. It lacked mitogenic and HIV-1 reverse transcriptase inhibiting activities, but exerted antiproliferative activity toward leukemia L1210 cells. [source] Characterization of the major allergens of Pachycondyla chinensis in ant sting anaphylaxis patientsCLINICAL & EXPERIMENTAL ALLERGY, Issue 4 2009E. K. Lee Summary Background The ant species Pachycondyla chinensis, which has spread from Far Eastern Asia to New Zealand and North America, induces anaphylactic reactions in human with its sting. However, the major allergens of P. chinensis have not yet been characterized. Methods We selected seven patients with histories of anaphylaxis induced by P. chinensis. Two-dimensional electrophoresis (2-DE) was used to identify the major allergens. We subsequently performed Western blots for P. chinensis -specific IgEs, N-terminal amino acid sequencing, ESI-MS/MS, and RT-PCR using primers based on the N-terminal sequence. Results Six of the anaphylactic subjects had an IgE specific to a 23 kDa allergen of P. chinensis. Two candidates for major allergens, 23 kDa (pI 8.7) and 25 kDa (pI 6.2), were revealed by 2-DE using P. chinensis -specific IgE immunoblotting. In N-terminal sequencing and ESI-MS/MS analysis, 23 kDa (pI 8.7) and 25 kDa (pI 6.2) allergens, belonging to the protein families of antigen 5, were identified and share marked amino acid sequence similarity. The 23 kDa allergen is 206 amino acids in length and homology searches showed 54.0% and 50.0% homology with Sol i 3 and Ves v 5, respectively. Conclusion The major allergens of P. chinensis are 23 kDa (pI 8.7) and 25 kDa (pI 6.2) proteins that belong to the antigen 5 family of proteins. [source] Structural characterization of the N-terminal mineral modification domains from the molluscan crystal-modulating biomineralization proteins, AP7 and AP24BIOPOLYMERS, Issue 5 2004Brandon A. Wustman Abstract The AP7 and AP24 proteins represent a class of mineral-interaction polypeptides that are found in the aragonite-containing nacre layer of mollusk shell (H. rufescens). These proteins have been shown to preferentially interfere with calcium carbonate mineral growth in vitro. It is believed that both proteins play an important role in aragonite polymorph selection in the mollusk shell. Previously, we demonstrated the 1,30 amino acid (AA) N-terminal sequences of AP7 and AP24 represent mineral interaction/modification domains in both proteins, as evidenced by their ability to frustrate calcium carbonate crystal growth at step edge regions. In this present report, using free N-terminal, C, -amide "capped" synthetic polypeptides representing the 1,30 AA regions of AP7 (AP7-1 polypeptide) and AP24 (AP24-1 polypeptide) and NMR spectroscopy, we confirm that both N-terminal sequences possess putative Ca (II) interaction polyanionic sequence regions (2 × ,DD, in AP7-1, ,DDDED, in AP24-1) that are random coil-like in structure. However, with regard to the remaining sequences regions, each polypeptide features unique structural differences. AP7-1 possesses an extended ,-strand or polyproline type II-like structure within the A11,M10, S12,V13, and S28,I27 sequence regions, with the remaining sequence regions adopting a random-coil-like structure, a trait common to other polyelectrolyte mineral-associated polypeptide sequences. Conversely, AP24-1 possesses random coil-like structure within A1,S9 and Q14,N16 sequence regions, and evidence for turn-like, bend, or loop conformation within the G10,N13, Q17,N24, and M29,F30 sequence regions, similar to the structures identified within the putative elastomeric proteins Lustrin A and sea urchin spicule matrix proteins. The similarities and differences in AP7 and AP24 N-terminal domain structure are discussed with regard to joint AP7,AP24 protein modification of calcium carbonate growth. © 2004 Wiley Periodicals, Inc. Biopolymers 2004 [source] Expression of antibodies using single-open reading frame vector design and polyprotein processing from mammalian cellsBIOTECHNOLOGY PROGRESS, Issue 3 2009Yune Z. Kunes Abstract We describe a novel polyprotein precursor-based approach to express antibodies from mammalian cells. Rather than expressing heavy and light chain proteins from separate expression units, the antibody heavy and light chains are contained in one single-open reading frame (sORF) separated by an intein gene fused in frame. Inside mammalian cells this ORF is transcribed into a single mRNA, and translated into one polypeptide. The antibody heavy and light chains are separated posttranslationally, assembled into the functional antibody molecule, and secreted into culture medium. It is demonstrated that Pol I intein from P. horikoshii mediates protein splicing and cleavage reactions in mammalian cells, in the context of antibody heavy and light chain amino acid sequences. To allow the separation of antibody heavy chain, light chain, and the intein, we investigated a number of intein mutations designed to inhibit intein-mediated splicing but preserve cleavage reactions. We have also designed constructs in which the signal peptide downstream from intein has altered hydrophobicity. The use of some of these mutant constructs resulted in more efficient antibody secretion, highlighting areas that can be further explored in improving such an expression system. An antibody secreted using one of the sORF constructs was characterized. This antibody has correct N-terminal sequences for both of its heavy and light chains, correct heavy and light chain MW as well as intact MW as measured by mass spectrometry. Its affinity to antigen, as measured by surface plasmon resonance (SPR), is indistinguishable from that of the same antibody produced using conventional method. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Enterohaemorrhagic Escherichia coli Tir requires a C-terminal 12-residue peptide to initiate EspFU -mediated actin assembly and harbours N-terminal sequences that influence pedestal lengthCELLULAR MICROBIOLOGY, Issue 9 2006Kenneth G. Campellone Summary Enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) both utilize type III secretion systems that translocate the effector protein Tir into the plasma membrane of mammalian cells in order to stimulate localized actin assembly into ,pedestals'. The Tir molecule that EPEC delivers is phosphorylated within its C-terminus on tyrosine-474, and a clustered 12-residue phosphopeptide encompassing this residue initiates an efficient signalling cascade that triggers actin polymerization. In addition to Y474, tyrosine-454 of EPEC Tir is phosphorylated, although inefficiently, and promotes actin polymerization at low levels. In contrast to EPEC Tir, EHEC Tir lacks Y474 and triggers pedestal formation in a phosphotyrosine-independent manner by interacting with an additional effector protein, EspFU. To identify EHEC Tir sequences that regulate localized actin assembly, we circumvented the strict requirements for type III translocation and directly expressed Tir derivatives in mammalian cells by transfection. Infection of Tir-expressing cells with a Tir-deficient EHEC strain demonstrated that ectopically expressed Tir localizes to the plasma membrane, is modified by mammalian serine-threonine kinases and is fully functional for actin pedestal formation. Removal of portions of the cytoplasmic N-terminus of Tir resulted in the generation of abnormally long pedestals, indicating that this region of EHEC Tir influences pedestal length. In the presence of the entire N-terminal domain, a 12-residue peptide from the C-terminus of EHEC Tir is both necessary and sufficient to recruit EspFU and initiate actin pedestal formation. This peptide encompasses the portion of EHEC Tir analogous to the EPEC Tir-Y454 region and is present within the Tir molecules of all pedestal-forming bacteria, suggesting that this sequence harbours a conserved signalling function. [source] | ||||||||||||||||