Identification and cloning of an aspartyl proteinase from Coccidioides immitis

A 45 kDa protein was isolated from a soluble vaccine prepared from formaldehyde-killed spherules of Coccidioides immitis. From the N-terminal amino acid sequence, the protein yielded a 17-amino-acid peptide that was homologous to sequences of other fungal aspartyl proteinases. The coccidioidal cDNA encoding the proteinase was amplified using oligonucleotide primers designed from the 45 kDa N-terminal amino acid sequence and a fungal aspartyl proteinase consensus amino acid sequence. The PCR product was cloned and sequenced, and the remaining 5' upstream and 3' downstream cDNA was amplified, cloned, and sequenced. The cDNA encoding the coccidioidal aspartyl proteinase open reading frame was cloned and the fusion protein containing a C-terminal His-tag expressed in E. coli. The recombinant aspartyl proteinase was purified by immobilized metal affinity chromatography. This recombinant protein will be used for further studies to evaluate its antigenicity, including protective immunogenicity.     

The primary structure of the Cytisus scoparius seed lectin and a carbohydrate-binding peptide.

The complete amino acid sequence of 2-acetamido-2-deoxy-D-galactose-binding Cytisus scoparius seed lectin II (CSII) was determined using a protein sequencer. After digestion of CSII with endoproteinase Lys-C or Asp-N, the resulting peptides were purified by reversed-phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. Comparison of the complete amino acid sequence of CSII with the sequences of other leguminous seed lectins revealed regions of extensive homology. The amino acid residues of concanavalin A (Con A) involved in the metal binding site are highly conserved among those of CSII. A carbohydrate-binding peptide of CSII was obtained from the endoproteinase Asp-N digest of CSII by affinity chromatography on a column of GalNAc-Gel. This peptide was retained on the GalNAc-Gel column and was presumed to have affinity for the column. The amino acid sequence of the retarded peptide was determined using a protein sequencer. The retarded peptide was found to correspond to the putative metal-binding region of Con A. These results strongly suggest that this peptide represents the carbohydrate-binding and metal ion-binding sites of CSII.     

Delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Aspergillus nidulans. Molecular characterization of the acvA gene encoding the first enzyme of the penicillin biosynthetic pathway

The Aspergillus nidulans gene (acvA) encoding the first catalytic steps of penicillin biosynthesis that result in the formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), has been positively identified by matching a 15-amino acid segment of sequence obtained from an internal CNBr fragment of the purified amino-terminally blocked protein with that predicted from the DNA sequence. acvA is transcribed in the opposite orientation to ipnA (encoding isopenicillin N synthetase), with an intergenic region of 872 nucleotides. The gene has been completely sequenced at the nucleotide level and found to encode a protein of 3,770 amino acids (molecular mass, 422,486 Da). Both fast protein liquid chromatography and native gel estimates of molecular mass are consistent with this predicted molecular weight. The enzyme was identified as a glycoprotein by means of affinity blotting with concanavalin A. No evidence for the presence of introns within the acvA gene has been found. The derived amino acid sequence of ACV synthetase (ACVS) contains three homologous regions of about 585 residues, each of which displays areas of similarity with (i) adenylate-forming enzymes such as parsley 4-coumarate-CoA ligase and firefly luciferase and (ii) several multienzyme peptide synthetases, including bacterial gramicidin S synthetase 1 and tyrocidine synthetase 1. Despite these similarities, conserved cysteine residues found in the latter synthetases and thought to be essential for the thiotemplate mechanism of peptide biosynthesis have not been detected in the ACVS sequence. These observations, together with the occurrence of putative 4'-phosphopantetheine-attachment sites and a putative thioesterase site, are discussed with reference to the reaction sequence leading to production of the ACV tripeptide. We speculate that each of the homologous regions corresponds to a functional domain that recognizes one of the three substrate amino acids.     

Isolation and partial sequencing of a pancreatic polypeptide-like neuropeptide from the liver fluke, Fasciola hepatica.

1. A pancreatic polypeptide (PP)-immunoreactive neuropeptide has been isolated and partially sequenced from the liver fluke, Fasciola hepatica. 2. Gel permeation chromatography of an acid ethanol extract of cattle flukes showed that the peptide is similar in size to mammalian (bovine) PP. 3. The Fasciola peptide was purified to homogeneity by means of reverse-phase HPLC, employing different column chemistries. 4. The purified peptide was sequenced using automated gas-phase Edman degradation and the first 24 amino acid residues determined.     

Isolation, functional, and partial biochemical characterization of galatrox, an acidic lectin from Bothrops atrox snake venom

Snake venom lectins have been studied in regard to their chemical structure and biological functions. However, little is known about lectins isolated from Bothrops atrox snake venom. We report here the isolation and partial functional and biochemical characterization of an acidic glycan-binding protein called galatrox from this venom. This lectin was purified by affinity chromatography using a lactosyl-sepharose column, and its homogeneity and molecular mass were evaluated by high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The purified galatrox was homogeneous and characterized as an acidic protein (pI 5.2) with a monomeric and dimeric molecular mass of 16.2 and 32.5 kDa, respectively. Alignment of N-terminal and internal amino acid sequences of galatrox indicated that this protein exhibits high homology to other C-type snake venom lectins. Galatrox showed optimal hemagglutinating activity at a concentration of 100 μg/ml and this effect was drastically inhibited by lactose, ethylenediaminetetraacetic acid, and heating, which confirmed galatrox's lectin activity. While galatrox failed to induce the same level of paw edema or mast cell degranulation as B. atrox crude venom, galatrox did alter cellular viability, which suggested that galatrox might contribute to venom toxicity by directly inducing cell death.     

Hyphenation of multi-dimensional chromatography and mass spectrometry for the at-line-analysis of the integrity of recombinant protein drugs

A robust tool is proposed for the rapid at-line verification of the identity and integrity of (recombinant) proteins, namely the hyphenation of multidimensional chromatography and mass spectrometry (MS). A recombinant human antibody produced in Chinese hamster ovary cells is taken as pertinent example. The recombinant human antibody is first captured from the production environment by affinity chromatography (rProtein A, isolation/concentration of the target molecule) and automatically transferred to an enzyme reactor (immobilized trypsin column) for digestion, thereby yielding different peptides corresponding to the protein sequence. The peptides are then separated on a reversed-phase column before being analyzed and identified by MS. This step does not require a fine resolution since the mass spectrometer can identify a variety of substances at the same time. The results are then analyzed in silico with suitable bio-informatic tools. When the gene sequence of the protein product is known, proteolytic cleavages can be predicted and the exact mass and hence the amino acid sequence of each peptide can thereby be deduced. Fitting experimental data and reference peptide sequences then provides important information about the integrity of the protein and more particularly about its sequence. In our case, the integrity of 45% of the light and 75% of the heavy chain sequences of the antibody could be verified within minutes.     

Semi-preparative HPLC purification of ribosomal proteins from Bacillus stearothermophilus and sequence determination of the highly conserved protein S19

Several proteins from the Bacillus stearothermophilus 30S ribosomal subunit which could not be isolated by conventional open-column chromatography were purified by high-performance liquid chromatography using a semi-preparative reverse-phase C4 column. Protein S19 was purified by this technique and the complete amino acid sequence determined. Protein S19 was fragmented and the peptides isolated in picomole quantities were sequenced by an improved manual 4-N,N-dimethylaminoazobenzene-4'-isothiocyanate (DABITC) technique; the presence of five consecutive C-terminal lysines in the S19 sequence was confirmed by gas-phase sequencing and fast-atom-bombardment (FAB) mass spectrometry. Protein S19 is composed of 91 amino acid residues which correspond to a molecular mass of 10,428 Da. 71% of the B. stearothermophilus S19 sequence was found to be identical with the corresponding ribosomal protein from Escherichia coli [Yaguchi and Wittmann (1978), FEBS Lett. 88, 227] and both sequences can be aligned without gaps. Among the known 26 amino acid sequences of the B. stearothermophilus and E. coli ribosome such a high degree of conservation has only been observed for a few proteins, all of which are known to be involved in the protein biosynthesis process. Although a clear function has not yet been assigned to protein S19, its high sequence conservation in these two eubacteria clearly indicates an important role of this protein for the function of the ribosome.     

Purification and characterization of a novel antimicrobial peptide from Brevibacillus laterosporus strain A60

A novel antimicrobial peptide, with molecular mass of 1602.0469Da, produced by Brevibacillus laterosporus strain A60 was isolated and purified from the soil of mango plants. The purification procedure consisted of ammonium sulfate precipitation, cation exchange chromatography on an HiTrap SP HP column, thin layer chromatography and High Performance Liquid Chromatography (HPLC) on C18 reversed-phase column. After the four isolation procedures, one peptide with antimicrobial activity was obtained and named BL-A60. The determination of the complete amino acid sequences of this peptide showed that it contains eleven amino acid residues, L-Y-K-L-V-K-V-V-L-N-M, and a choline connected to the N-terminal and a tenuazonic acid modified of the C-terminal. This peptide shows relatively low identification to other antimicrobial peptides from bacteria. Purified BL-A60 showed high pH and thermal stability and a strong inhibition of different stages of the life cycle of Phytophthora capsici, including mycelial growth, sporangia formation and cystospore germination, with EC(50) values of 7.89, 0.60 and 21.96 μg ml(-1), respectively.     

Purification and characterization of a surface protein from Lactobacillus fermentum 104R that binds to porcine small intestinal mucus and gastric mucin

An adhesion-promoting protein involved in the binding of Lactobacillus fermentum strain 104R to small intestinal mucus from piglets and to partially purified gastric mucin was isolated and characterized. Spent culture supernatant fluid and bacterial cell wall extracts were fractionated by ammonium sulfate precipitation and gel filtration. The active fraction was purified by affinity chromatography. The adhesion-promoting protein was detected in the fractions by adhesion inhibition and dot blot assays and visualized by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate-PAGE, and Western blotting with horseradish peroxidase-labeled mucus and mucin. The active fraction was characterized by estimating the relative molecular weight and by assessing the presence of carbohydrates in, and heat sensitivity of, the active region of the adhesion-promoting protein. The purified protein was digested with porcine trypsin, and the peptides were purified in a SMART system. The peptides were tested for adhesion to horseradish peroxidase-labeled mucin by using the dot blot adhesion assay. Peptides which bound mucin were sequenced. It was shown that the purified adhesion-promoting protein on the cell surface of L. fermentum 104R is extractable with 1 M LiCl and low concentrations of lysozyme but not with 0.2 M glycine. The protein could be released to the culture supernatant fluid after 24 h of growth and had affinity for both small intestinal mucus and gastric mucin. In the native state this protein was variable in size, and it had a molecular mass of 29 kDa when denatured. The denatured protein did not contain carbohydrate moieties and was not heat sensitive. Alignment of amino acids of the adhering peptides with sequences deposited in the EMBL data library showed poor homology with previously published sequences. The protein represents an important molecule for development of probiotics.     

Expression, purification, and characterisation of nesiritide using an E. coli expression system

Nesiritide, the recombinant human brain natriuretic peptide, is involved in the regulation of cardiovascular homeostasis and has been approved for treatment of patients with congestive heart failure. We prepared a synthetic cDNA construct of Nesiritide to generate a fusion protein with an affinity handle and 41 amino acid peptide of β-galactosidase. The fusion protein was expressed mainly in the inclusion bodies and accounted for approximately 20% of total cellular protein. After purification by Ni-IDA affinity chromatography and renaturation, the fusion protein was cleaved with purified recombinant enterokinase. Nesiritide was purified by pH precipitation/ion exchange chromatography followed by source phenyl chromatography to obtain protein with > 99% purity (determined by RPHPLC) and a mass of 3,464 Daltons. The potency (ED₅₀) of the purified protein was equivalent to that of Natrecor (Innovator formulation). Analytical methods were developed to identify oxidised, reduced and other related impurities. The expression strategy described in this work allows the convenient generation of high yield Nesiritide and enabled ease of purification.     

Affinity purification of the C alpha and C beta isoforms of the catalytic subunit of cAMP-dependent protein kinase

A synthetic peptide of 18 amino acids corresponding to the inhibitory domain of the heat-stable protein kinase inhibitor was synthesized and shown to inhibit both the C alpha and C beta isoforms of the catalytic (C) subunit of cAMP-dependent protein kinase. Extracts from cells transfected with expression vectors coding for the C alpha or the C beta isoform of the C subunit required 200 nM protein kinase inhibitor peptide for half-maximal inhibition of kinase activity in extracts from these cells. An affinity column was constructed using this synthetic peptide, and the column was incubated with protein extracts from cells overexpressing C alpha or C beta. Elution of the affinity column with arginine allowed single step isolation of purified C alpha and C beta subunits. The C alpha and C beta proteins were enriched 200-400-fold from cellular extracts by this single step of affinity chromatography. No residual inhibitory peptide activity could be detected in the purified protein. The purified C subunit isoforms were used to demonstrate preferential antibody reactivity with the C alpha isoform by Western blot analysis. Furthermore, preliminary characterization showed both isoforms have similar apparent Km values for ATP (4 microM) and for Kemptide (5.6 microM). These results demonstrate that a combination of affinity chromatography employing peptides derived from the heat-stable protein kinase inhibitor protein and the use of cells overexpressing C subunit related proteins may be an effective means for purification and characterization of the C subunit isoforms. Furthermore, this method of purification may be applicable to other kinases which are known to be specifically inhibited by small peptides.     

Biochemical characterization, cDNA cloning and protein crystallization of aryl-alcohol oxidase from Pleurotus pulmonarius

Aryl-alcohol oxidase (AAO) involved in lignin degradation by Pleurotus pulmonarius has been purified and characterized. The enzyme was produced in glucose-peptone medium and isolated in a sole chromatographic step using Sephacryl S-200. The purified enzyme is an extracellular glycoprotein with 14% N-carbohydrate content and an estimated molecular mass of 70.5 kDa and pI of 3.95. The kinetic studies showed the highest enzyme affinity against p-anisyl alcohol, with constants similar to those of Pleurotus eryngii and Bjerkandera adusta AAO but different from the intracellular AAO described in Phanerochaete chrysosporium, which present the highest activity on m-anisyl alcohol. Simultaneously, the cDNA of P. pulmonarius AAO has been cloned and sequenced. The translation of this sequence consisted of 593 amino acids including a signal peptide of 27 amino acids. The comparison with other alcohol oxidases, 35% amino acid identity with glucose oxidase, showed highly conserved amino acid sequences in N-terminal and C-terminal regions, in spite of differences in substrate specificity. Crystallization of AAO, carried out for the first time using the P. pulmonarius enzyme, will permit to obtain a molecular model for this oxidase and establish some characteristic of its catalytic site and general structure.     

The amino-acid sequence of isoinhibitor K form snails (Helix pomatia). A sequence determination by automated Edman degradation and mass-spectral identification of the phenylthiohydantoins.

Performic-acid-oxidized isoinhibitor K of snails (Helix pomatia) was subjected to arginine-directed tryptic proteolysis. Six peptide fragments including one overlap peptide from limited cleavage of the Arg-3-Pro-4 bond were purified to homogeneity. Four arginine peptides and the C-terminal peptide were sequenced by automatic Edman degradation using a special peptide program. The phenylthiohydantoins were all identified by chemical ionization mass spectrometry, except four cysteic acid residues that were identified on an amino acid analyzer after acid hydrolysis. Quantitative evaluation of the phenylthiohydantoins by chemical ionization mass spectrometry using total molecular-ion beam integration greatly facilitated sequencing. The mass spectrum of the dipeptide less than Glu-Gly revealed that the N-terminus was blocked by pyroglutamic acid. The complete amino acid sequence of isoinhibitor K was determined. An almost 50% homology between the sequences of the snail inhibitor and the bovine trypsin-kallikrein inhibitor (Kunitz) became obvious. A comparison of all homologous sequences of this particular class of proteins known to date is presented.     

Association of annexin V with prolactin in the rat anterior pituitary gland.

When pituitary extracts were subjected to non denaturing polyacrylamide gel electrophoresis, an unknown protein was found to associate with a proportion of the prolactin. This protein was dissociated from prolactin by sodium dodecyl sulfate. The protein was purified and sequenced. As the amino terminus was blocked, the amino acid sequences of three peptide fragments were determined. The obtained sequences of 41 amino acids were identical to partial sequences of a known protein, rat Annexin V. The molecular mass, 36 kDa, was also the same as the molecular weight of Annexin V. The existence of Annexin V mRNA in rat pituitary glands was also confirmed by polymerase chain reaction. These results show that Annexin V, a member of the calcium-dependent phospholipid binding proteins, is synthesized in the rat pituitary gland, and suggest its association with prolatin in the gland.     

The cDNA sequence of mouse Pgp-1 and homology to human CD44 cell surface antigen and proteoglycan core/link proteins

We describe the isolation and sequencing of a cDNA encoding mouse Pgp-1. An oligonucleotide probe corresponding to the NH2-terminal sequence of the purified protein was synthesized by the polymerase chain reaction and used to screen a mouse macrophage lambda gt11 library. A cDNA clone with an insert of 1.2 kilobases was selected and sequenced. In Northern blot analysis, only cells expressing Pgp-1 contained mRNA species that hybridized with this Pgp-1 cDNA. The nucleotide sequence of the cDNA has a single open reading frame that yields a protein-coding sequence of 1076 base pairs followed by a 132-base pair 3'-untranslated sequence that includes a putative polyadenylation signal but no poly(A) tail. The translated sequence comprises a 13-amino acid signal peptide followed by a polypeptide core of 345 residues corresponding to an Mr of 37,800. Portions of the deduced amino acid sequence were identical to those obtained by amino acid sequence analysis from the purified glycoprotein, confirming that the cDNA encodes Pgp-1. The predicted structure of Pgp-1 includes an NH2-terminal extracellular domain (residues 14-265), a transmembrane domain (residues 266-286), and a cytoplasmic tail (residues 287-358). Portions of the mouse Pgp-1 sequence are highly similar to that of the human CD44 cell surface glycoprotein implicated in cell adhesion. The protein also shows sequence similarity to the proteoglycan tandem repeat sequences found in cartilage link protein and cartilage proteoglycan core protein which are thought to be involved in binding to hyaluronic acid.     

Molecular Cloning and Expression of a Mouse Brain cDNA Encoding a Novel Protein Target of Calcyclin

Abstract: A protein target of mouse calcyclin, p30, which we call calcyclin-binding protein (CacyBP), was identified in mouse brain and Ehrlich ascites tumor (EAT) cells. The amino acid sequence of the CacyBP chymotryptic peptide was used to prepare synthetic oligonucleotides that served as a probe to screen the mouse brain cDNA library. A 1.4-kb positive clone was detected, isolated, and sequenced. The analyzed clone contains an open reading frame encoding a protein of a molecular mass of ～26 kDa. The nucleotide and predicted amino acid sequences indicate that CacyBP is a novel protein. The results obtained from northern blots show that the CacyBP gene is expressed predominantly in mouse brain and EAT cells. Using a pGEX vector the recombinant CacyBP was expressed in Escherichia coli, and its properties were analyzed. The recombinant protein interacts with calcyclin at a physiologically relevant range of Ca²⁺ in solution during affinity chromatography and on blots. Because CacyBP, like calcyclin, is present in the brain, the interaction of these two proteins might be involved in calcium signaling pathways in neuronal tissue.     

Isolation and partial sequencing of a pancreatic polypeptide-like neuropeptide from the liver fluke,Fasciola hepatica

1. A pancreatic polypeptide (PP)-immunoreactive neuropeptide has been isolated and partially sequenced from the liver fluke, Fasciola hepatica.2. Gel permeation chromatography of an acid ethanol extract of cattle flukes showed that the peptide is similar in size to mammalian (bovine) PP.3. The Fasciola peptide was purified to homogeneity by means of reverse-phase HPLC, employing different column chemistries.4. The purified peptide was sequenced using automated gas-phase Edman degradation and the first 24 amino acid residues determined.     

Isolation of a cDNA coding for human galactosyltransferase

Human milk galactosyltransferase (EC 2.4.1.22) was purified to homogeneity using affinity chromatography. Edman degradation was used to determine the amino acid sequences of eight peptide fragments isolated from the purified enzyme. A 60-mer "optimal" oligonucleotide probe that corresponded to the amino acid sequence of one of the galactosyltransferase peptide fragments was constructed and used to screen a lambda gt10 cDNA library. Two hybridization-positive recombinant phages, each with a 1.7 Kbp insert, were detected among 3 X 10(6) recombinant lambda gt10 phages. Sequencing of one of the cDNA inserts revealed a 783 bp galactosyltransferase coding sequence. The remainder of the sequence corresponded to the 3'-region of the mRNA downstream from the termination codon.     

应用细菌鞭毛递呈的随机肽库研究丙型肝炎病毒核心蛋白B细胞抗原表位

设计了利用大扬杆菌鞭毛蛋白递呈的随机十二肽库研究ＨＣＶ核心蛋白Ｂ细胞抗原位的实验程序：１利用大肠杆有达质粒ｐＱＥ－３０有达并纯化ＨＣＶ核心蛋白Ｐ１９;２利用Ｐ１９蛋白亲和层析纯化ＨＣＶ感染者血清的抗ＨＣＶ核心蛋白多克隆抗体;     

Purification and characterization of placental protein 5

This report describes the purification of placental protein 5, PP5, from the human placenta by two affinity chromatography steps, the first with Heparin-Sepharose and the second with Sepharose-linked monoclonal anti-PP5 antibody. The final purification is achieved by reversed-phase high performance liquid chromatography. In SDS-polyacrylamide gel electrophoresis under reducing or nonreducing conditions, PP5 purified in this study migrates as one major band at 36 kD. The previously purified PP5 is more heterogeneous: under nonreducing conditions it migrates at 30 kD and, after reduction, it gives three bands at 16.8 kD, 18.3 kD, and 19.0 kD. In Western blot analysis, both purified proteins react with polyclonal and monoclonal anti-PP5 antibodies. Three N-terminal amino acid sequences are obtained for the previously purified PP5, whereas the N-terminal of PP5 purified in this study is blocked. These results suggest that PP5 previously purified in the absence of protease inhibitors, does not represent the native form of PP5. Computer comparison of the obtained amino acid sequences revealed no significant homology to known protein sequences.