RESUMEN
The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and executes interlaboratory studies investigating the use of automated Edman degradation for protein and peptide analysis. In 2008, the ESRG enlisted the help of core sequencing facilities to investigate the effects of a repeating amino acid tag at the N-terminus of a protein. Commonly, to facilitate protein purification, an affinity tag containing a polyhistidine sequence is conjugated to the N-terminus of the protein. After expression, polyhistidine-tagged protein is readily purified via chelation with an immobilized metal affinity resin. The addition of the polyhistidine tag presents unique challenges for the determination of protein identity using Edman degradation chemistry. Participating laboratories were asked to sequence one protein engineered in three configurations: with an N-terminal polyhistidine tag; with an N-terminal polyalanine tag; or with no tag. Study participants were asked to return a data file containing the uncorrected amino acid picomole yields for the first 17 cycles. Initial and repetitive yield (R.Y.) information and the amount of lag were evaluated. Information about instrumentation and sample treatment was also collected as part of the study. For this study, the majority of participating laboratories successfully called the amino acid sequence for 17 cycles for all three test proteins. In general, laboratories found it more difficult to call the sequence containing the polyhistidine tag. Lag was observed earlier and more consistently with the polyhistidine-tagged protein than the polyalanine-tagged protein. Histidine yields were significantly less than the alanine yields in the tag portion of each analysis. The polyhistidine and polyalanine protein-R.Y. calculations were found to be equivalent. These calculations showed that the nontagged portion from each protein was equivalent. The terminal histidines from the tagged portion of the protein were demonstrated to be responsible for the high lag during N-terminal sequence analysis.
Asunto(s)
Marcadores de Afinidad/análisis , Histidina/análisis , Hormona de Crecimiento Humana/química , Péptidos/análisis , Análisis de Secuencia de Proteína/métodos , Secuencia de Aminoácidos , Aminoácidos/aislamiento & purificación , Automatización , Eficiencia , Hormona de Crecimiento Humana/aislamiento & purificación , Humanos , Laboratorios/normas , Datos de Secuencia Molecular , Compuestos Organofosforados , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Análisis de Secuencia de Proteína/instrumentación , Análisis de Secuencia de Proteína/normas , TransfecciónRESUMEN
Evidence for a key role of beta-amyloid (Abeta) in Alzheimer's disease has led to considerable interest in potential therapeutic strategies targeting enzymes involved in processing the amyloid precursor protein (APP). Beta-site APP Cleaving Enzyme (BACE or beta-secretase) is a membrane bound aspartyl protease that has been shown to be directly involved in Abeta production and, therefore, is at the forefront of therapeutic targets in the treatment of Alzheimer's disease. BACE-2, an enzyme closely related to BACE, regulates Abeta production in a manner antagonistic to BACE, suggesting that non-selective inhibition of BACE-2 by BACE inhibitors might impair the lowering of Abeta. The design of BACE inhibitors that do not inhibit BACE-2 would be enhanced by structural and kinetic studies, efforts that typically demand considerable amounts of both enzymes. A BACE-2 construct containing 19 residues of the BACE prosegment followed by the BACE-2 catalytic domain sequence, Asp36-Trp447, was produced in E. coli inclusion bodies (IB) at 110-140 mg/L cell culture. Exploration of a variety of refolding conditions resulted in an efficient method for refolding the resulting pro-BACE-2 construct, and this protein undergoes facile autocatalytic cleavage, optimal at pH 4, at the Leu40- downward arrow-Ala41 bond. Refolded BACE-2 was purified by anion exchange, molecular sieve, and affinity chromatographies, yielding 105 mg of homogeneous enzyme (kcat/ Km = 1.2 x 10(4) x M(-1) x sec(-1)) from 8 liters of E. coli cell culture.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/química , Ácido Aspártico Endopeptidasas/química , Dominio Catalítico/genética , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Cromatografía de Afinidad , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Cuerpos de Inclusión/metabolismo , Datos de Secuencia Molecular , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de Proteína , TemperaturaRESUMEN
BACE, or beta-secretase, is an attractive target in the treatment of Alzheimer's Disease because of its involvement in the generation of amyloid beta peptides. BACE is a type I transmembrane aspartyl protease composed of pre-, pro-, catalytic, transmembrane and cytoplasmic domains. For the present study, the coding sequence was truncated just before the transmembrane domain and the resulting construct was extended with the C-terminal addition of a (His)(6) and expressed in several mammalian host cells. The enzyme expressed in CHO cells had the best crystallographic behavior and was purified in large quantities in a three step procedure. The purified BACE was comprised of two forms, namely the full length proBACE construct beginning with Thr(1), and a derivative missing the first 24 amino acids beginning with E(25). These BACE precursors co-crystallized in the presence of inhibitors yielding structures to 3.2 A resolution. HIV-1 protease treatment of this mixture resulted in complete cleavage of the F(39)-V(40) bond, leaving the V(40)EM...ES(432) (His)(6) derivative that was purified yielding an enzyme that was no more active than untreated BACE but co-crystallized with inhibitors producing well shaped, bipyramidal co-crystals diffracting to 2.6 A resolution.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/aislamiento & purificación , Ácido Aspártico Endopeptidasas/aislamiento & purificación , Proteasa del VIH/metabolismo , Precursores de Proteínas/aislamiento & purificación , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Células CHO , Cricetinae , Cricetulus , Cristalización , Humanos , Datos de Secuencia Molecular , Estructura Molecular , Conformación Proteica , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Análisis de Secuencia de Proteína , Difracción de Rayos XRESUMEN
BACE (beta-site APP cleaving enzyme) or beta-secretase, the enzyme responsible for processing APP to give the N-terminal portion of the Abeta peptide, is a membrane bound aspartyl protease consisting of an ectodomain catalytic unit, a C-terminal transmembrane segment and a cytoplasmic domain. Three BACE constructs, pET11a-BACE, pQE80L-BACE, and pQE70-BACE were designed to terminate at a position just before the transmembrane domain (Ser(432)) and are described schematically below. (1) pET11a-T7.Tag-G-S-M-(A-8GV......QTDES(432)), (2) pQE80L-Met-R-G-S-(His)(6)-G-S-I-E-T-D-(T(1)QH...QTDES(432)), and (3) pQE70-Met-BACE (R(36)GSFVEMG....PQTDES(432) (His) (6)) Each construct was over-expressed in Escherichia coli as inclusion bodies. The inclusion body proteins were solubilized in urea and refolded by dilution in water to yield active enzyme. Maximal activity for pET11a-BACE and pQE80L-BACE was usually reached at day 3 to 4, while construct pQE70-BACE required about 21 days. Active BACE was purified to homogeneity by anion-exchange chromatography and affinity chromatography over a column of immobilized peptide inhibitor. The process, easily scalable to 60 liters of cell culture, yielded in excess of 400 mg of active enzyme for crystallographic analysis. Highly purified pET11a-BACE and pQE70-BACE formed complexes with various inhibitors, the latter protein giving crystals diffracting up to 1.45 A resolution. In addition, a crystal form that does not require the presence of an inhibitor has been obtained for pQE70-BACE. This ligand-free crystal form has proven useful for the preparation of BACE-inhibitor complexes in soaking experiments.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/aislamiento & purificación , Ácido Aspártico Endopeptidasas/aislamiento & purificación , Pliegue de Proteína , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Células CHO , Cricetinae , Cricetulus , Cristalización , Escherichia coli/genética , Escherichia coli/metabolismo , Proteasa del VIH/metabolismo , Humanos , Datos de Secuencia Molecular , Estructura Molecular , Conformación Proteica , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Difracción de Rayos XRESUMEN
The Edman Sequencing Research Group (ESRG) designs studies on the use of Edman degradation for protein and peptide analysis. These studies provide a means for participating laboratories to compare their analyses against a benchmark of those from other laboratories that provide this valuable service. The main purpose of the 2006 study was to determine how accurate Edman sequencing is for quantitative analysis of polypeptides. Secondarily, participants were asked to identify a modified amino acid residue, N-epsilon-acetyl lysine [Lys(Ac)], present within one of the peptides. The ESRG 2006 peptide mixture consisted of three synthetic peptides. The Peptide Standards Research Group (PSRG) provided two peptides, with the following sequences: KAQYARSVLLEKDAEPDILELATGYR (peptide B), and RQAKVLLYSGR (peptide C). The third peptide, peptide C*, synthesized and characterized by ESRG, was identical to peptide C but with acetyl lysine in position 4. The mixture consisted of 20% peptide B and 40% each of peptide C and its acetylated form, peptide C*. Participating laboratories were provided with two tubes, each containing 100 picomoles of the peptide mixture (as determined by quantitative amino acid analysis) and were asked to provide amino acid assignments, peak areas, retention times at each cycle, as well as initial and repetitive yield estimates for each peptide in the mixture. Details about instruments and parameters used in the analysis were also collected. Participants in the study with access to a mass spectrometer (MALDI-TOF or ESI) were asked to provide information about the relative peak areas of the peptides in the mixture as a comparison with the peptide quantitation results from Edman sequencing. Positive amino acid assignments were 88% correct for peptide C and 93% correct for peptide B. The absolute initial sequencing yields were an average of 67% for peptide (C+C*) and 65.6 % for peptide B. The relative molar ratios determined by Edman sequencing were an average of 4.27 (expected ratio of 4) for peptides (C+C*)/B, and 1.49 for peptide C*/C (expected ratio of 1); the seemingly high 49% error in quantification of Lys(Ac) in peptide C* can be attributed to commercial unavailability of its PTH standard. These values compare very favorably with the values obtained by mass spectrometry.
Asunto(s)
Péptidos/análisis , Análisis de Secuencia de Proteína , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Péptidos/química , Análisis de Secuencia de Proteína/instrumentación , Análisis de Secuencia de Proteína/normas , Análisis de Secuencia de Proteína/tendencias , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Identification of modified amino acids can be a challenging part for Edman degradation sequence analysis, largely because they are not included among the commonly used phenylthiohydantion amino acid standards. Yet many can have unique retention times and can be assigned by an experienced researcher or through the use of a guide showing their typical chromatography characteristics. The Edman Sequencing Research Group (ESRG) 2005 study is a continuation of the 2004 study, in which the participating laboratories were provided a synthetic peptide and asked to identify the modified amino acids present in the sequence. The study sample provided an opportunity to sequence a peptide containing a variety of modified amino acids and note their retention times relative to the common amino acids. It also allowed the ESRG to compile the chromatographic properties and intensities from multiple instruments and tabulate an average elution position for these modified amino acids on commonly used instruments. Participating laboratories were given 2000 pmoles of a synthetic peptide, 18 amino acids long, containing the following modified amino acids: dimethyl- and trimethyl-lysine, 3-methyl-histidine, N-carbamyl-lysine, cystine, N-methyl-alanine, and isoaspartic acid. The modified amino acids were interspersed with standard amino acids to help in the assessment of initial and repetitive yields. In addition to filling in an assignment sheet, which included retention times and peak areas, participants were asked to provide specific details about the parameters used for the sequencing run. References for some of the modified amino acid elution characteristics were provided and the participants had the option of viewing a list of the modified amino acids present in the peptide at the ESRG Web site. The ABRF ESRG 2005 sample is the seventeenth in a series of studies designed to aid laboratories in evaluating their abilities to obtain and interpret amino acid sequence data.
Asunto(s)
Aminoácidos/análisis , Aminoácidos/química , Análisis de Secuencia de Proteína , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Compuestos Organofosforados , Péptidos/química , Feniltiohidantoína/químicaRESUMEN
We report here the cloning and high-level expression of a soluble proform of human caspase 3 (Ser(24)-H(277)) engineered to contain a short stretch of N-terminal sequence (MTISDSPREQD) from the prosegment of procaspase 8 and a C-terminal heptahistidine tag. The precursor protein isolated from extracts of recombinant Escherichia coli by immobilized metal-ion affinity chromatography was predominantly unprocessed and migrated as a 32-kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gels. Incubation of this protein with recombinant human caspase 8 produced fragments characteristic of the properly processed caspase 3, but the product was inactive. Amino-terminal sequence analysis of the caspase 3 polypeptides proved that caspase 8 had specifically cleaved the Asp(175)-Ser(176) bond to yield the expected p18 and p12 subunits, with partial cleavage at the Asp(28)-Ser(29) bond to release the prosegment. The lack of caspase 3 activity was found to be the result of a fortuitous mutation in which Trp(206) in the S4 subsite was replaced by arginine (W206R). This mutant procaspase 3, which we call m-pro3, serves as a useful reagent with which to test the efficacy of caspase 8 inhibitors in blocking processing of the natural polypeptide substrate of this enzyme and may be valuable as a source of "proenzyme" for crystallographic analysis.
Asunto(s)
Caspasas/genética , Caspasas/metabolismo , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Arginina/genética , Caspasa 3 , Caspasa 8 , Caspasa 9 , Inhibidores de Caspasas , Caspasas/biosíntesis , Caspasas/aislamiento & purificación , Clonación Molecular , Dimerización , Activación Enzimática/genética , Inhibidores Enzimáticos/farmacología , Precursores Enzimáticos/biosíntesis , Precursores Enzimáticos/aislamiento & purificación , Escherichia coli/enzimología , Escherichia coli/genética , Vectores Genéticos , Humanos , Hidrólisis , Sustancias Macromoleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Especificidad por Sustrato/genética , Triptófano/genéticaRESUMEN
Receptor tyrosine kinases may use intrasteric inhibition to suppress autophosphorylation prior to growth factor stimulation. To test this hypothesis we made an Asp1161Ala mutant in the activation loop that relieved intrasteric inhibition of the unphosphorylated insulin receptor (IR) and its recombinant cytoplasmic kinase domain (IRKD) without affecting the activated state. Solution studies with the unphosphorylated mutant IRKD demonstrated conformational changes and greater catalytic efficiency from a 10-fold increase in k(cat) and a 15-fold-lower K(m ATP) although K(m peptide) was unchanged. Kinetic parameters of the autophosphorylated mutant and wild-type kinase domains were virtually identical. The Asp1161Ala mutation increased the rate of in vitro autophosphorylation of the IRKD or IR at low ATP concentrations and in the absence of insulin. However, saturation with ATP (for the IRKD) or the presence of insulin (for the IR) yielded equivalent rates of autophosphorylation for mutant versus wild-type kinases. Despite a biochemically more active kinase domain, the mutant IR expressed in C2C12 myoblasts was not constitutively autophosphorylated. However, it displayed a 2.5-fold-lower 50% effective concentration for insulin stimulation of autophosphorylation and was dephosphorylated more slowly following withdrawal of insulin than wild-type IR. In tests of the regulation of the unphosphorylated basal state, these results demonstrate that neither intrasteric inhibition against ATP binding nor suppression of kinase activity is required to prevent premature autophosphorylation of the IR. Finally, the lower rate of dephosphorylation suggests invariant residues of the activation loop such as Asp1161 may function at multiple junctures in cellular regulation of receptor tyrosine kinases.
Asunto(s)
Adenosina Trifosfato/metabolismo , Receptor de Insulina/metabolismo , Transducción de Señal/fisiología , Línea Celular , Medio de Cultivo Libre de Suero , Immunoblotting , Insulina/metabolismo , Cinética , Mutagénesis Sitio-Dirigida , Fosforilación , Conformación Proteica , Receptor de Insulina/química , Receptor de Insulina/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/genética , Espectrometría de FluorescenciaRESUMEN
A gene coding for a truncated form of human procaspase 8 has been cloned and expressed in Escherichia coli. This construct contains M(206) through D(479) of human procaspase 8, preceded by an N-terminal polyhistidine tag. The recombinant protein, containing 286 amino acids, was expressed in high yield in the form of inclusion bodies (IB). The IB were solubilized in guanidinium chloride and dialyzed against 50% acetic acid. The solution was mixed with 9 volumes of H(2)O and then rapidly diluted from the acidic medium to one containing 1.0 M Tris, pH 8.0, and 5 mM DTT. SDS-PAGE analysis of the soluble, dilute protein solution (20-30 microgram of protein/ml) showed a single 33-kDa band corresponding to the nonprocessed, inactive procaspase 8. Concentration of the dilute protein to levels as high as 2 mg/ml resulted in only modest (1-10%) autocatalytic conversion to the 19- and 11-kDa polypeptide subunits which are characteristic of the activated enzyme. Further concentration of these protein solutions to a near-dry state on the ultrafiltration membrane, followed by washing of the membrane with buffer, led to extracts containing high yields of enzyme showing a specific activity of 8.43 micromol/min/mg against the chromogenic substrate Ac-IETD-pNA. SDS-PAGE, protein sequencing, and mass spectrometric analysis of these extracts showed complete conversion of the 33-kDa procaspase 8 to the 19- and 11-kDa subunits of activated caspase 8. This method allows for preparation of 100-mg quantities of highly pure and active recombinant human caspase 8. Enzyme activity was shown to be associated with a heterotetrameric complex that is converted to an inactive dimer upon storage.
Asunto(s)
Caspasas/metabolismo , Precursores Enzimáticos/metabolismo , Secuencia de Aminoácidos , Caspasa 8 , Caspasa 9 , Caspasas/química , Caspasas/genética , Cromatografía Líquida de Alta Presión , Clonación Molecular , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Escherichia coli/enzimología , Escherichia coli/genética , Humanos , Cinética , Espectrometría de Masas , Datos de Secuencia Molecular , Peso Molecular , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Human platelet heparanase has been purified to homogeneity and shown to consist of two, non-covalently associated polypeptide chains of molecular masses 50 and 8 kDa. Protein sequencing provided the basis for determination of the full-length cDNA for this novel protein. Based upon this information and results from protein analysis and mass spectrometry, we propose a scheme to define the structural organization of heparanase in relation to its precursor forms, proheparanase and pre-proheparanase. The 8- and 50-kDa chains which make up the active enzyme reside, respectively, at the NH(2)- and COOH-terminal regions of the inactive precursor, proheparanase. The heparanase heterodimer is produced by excision and loss of an internal linking segment. This paper is the first to suggest that human heparanase is a two-chain enzyme.
Asunto(s)
Precursores Enzimáticos/metabolismo , Glucuronidasa , Glicósido Hidrolasas/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Plaquetas/enzimología , Cromatografía Líquida de Alta Presión , ADN Complementario , Dimerización , Activación Enzimática , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Humanos , Datos de Secuencia Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
In preclinical pharmacokinetic studies and in in vitro rat, dog, and human primary hepatocyte incubations, the sulfonamide (-NH-SO(2)-) bond of a potent inhibitor of the HIV-1 protease containing the p-cyanopyridinyl moiety (PNU-109112), undergoes metabolic cleavage to form the corresponding amine metabolite (PNU-143070). Strikingly, a compound, PNU-140690, obtained by substituting the cyanopyridinyl group of PNU-109112 with a trifluoropyridinyl moiety, was stable under the same in vivo and in vitro conditions used for PNU-109112. The apparent "sulfonamidase activity" present in liver was localized to the cytosolic fraction and shown to be an enzyme-mediated reaction requiring reduced glutathione (GSH). The enzyme responsible was purified in a single step on a GSH immobilized gel and was identified as glutathione-S-transferase (GST) by sequence analysis of peptides obtained by tryptic digestion of the purified protein. Moreover, a mixture of GST isoenzymes purified from rat liver, and three recombinant human GST isoforms, A1-1, M1-1, and P1-1, were active toward PNU-109112 sulfonamide cleavage; the three isoforms exhibited differential rates of PNU-109112 cleavage, demonstrating isoenzyme selectivity.
Asunto(s)
Diuréticos/metabolismo , Glutatión Transferasa/metabolismo , Sulfonamidas/metabolismo , Secuencia de Aminoácidos , Animales , Biotransformación , Línea Celular , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Perros , Electroforesis en Gel de Poliacrilamida , Glutatión Transferasa/química , Humanos , Hidrólisis , Isoenzimas/metabolismo , Masculino , Espectrometría de Masas , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Fracciones Subcelulares/metabolismoRESUMEN
Recombinant yeast ubiquitin C-terminal hydrolase (YUH1), which has an N-terminal (His)(6) tag, and an autolysis-resistant mutant of the human immunodeficiency virus-1 protease (HIV-1 Pr) have been used as specific proteases to yield peptides from a ubiquitin conjugate. In the present example, connective tissue-activating peptide (CTAPIII) and neutrophil-activating peptide 2 (NAP/2) were generated by digestion of a ubiquitin-CTAPIII conjugate with YUH1 and HIV Pr, respectively, as indicated below: [see text] YUH1 cleaved at the peptide bond formed by the C-terminal Gly(76) of ubiquitin (Ub) and the N-terminal Asn(1) of the 85-residue peptide CTAPIII. The HIV-1 Pr cleaved between Tyr(15) and Ala(16), the N-terminal Ala of the 70-residue peptide NAP/2. Both enzymes produced authentic peptides from the Ub fusion protein, with a nearly 100% yield. The liberated CTAPIII and NAP/2 were separated from (His)(6)-Ub, the trace amounts of unreacted (His)(6)-Ub-CTAPIII, HIV-1 Pr, and the (His)(6)-YUH1 by passage over a nickel-chelate column; the final yield was about 10 mg of peptide/liter of cell culture. (His)(6)-YUH1, the HIV Pr mutant, and the (His)(6)-Ub-CTAPIII substrate were all expressed individually in Escherichia coli. (His)(6)-YUH1 and (His)(6)-Ub-CTAPIII were highly expressed in a soluble form, but about 75% of the total (His)(6)-YUH1 was also found in inclusion bodies. Both proteins from the soluble fractions were easily purified in a single step by immobilized metal ion affinity chromatography with a yield of about 27 mg of (His)(6)-Ub-CTAPIII and 13.6 mg of (His)(6)-YUH1 protein/liter of cell culture. Chemotactic factor activity, as assessed by the neutrophil shape change assay, was observed for NAP/2, but not for CTAPIII. This strategy, which employs YUH1 and the HIV-1 Pr as tools for the highly selective cleavage of the chimeric substrate, should be applicable to the large-scale production of a variety of peptides.
Asunto(s)
Endopeptidasas/metabolismo , Proteasa del VIH/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía de Afinidad , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Proteínas Recombinantes/metabolismo , Ubiquitinas/metabolismo , beta-TromboglobulinaRESUMEN
Human cytomegalovirus protease (CMV PR) is a target for the development of antiviral therapeutics. To obtain large amounts of native protease, a 268-amino-acid polypeptide with a hexahistidinyl tag at the C terminus was expressed in Escherichia coli. The first 262 amino acids of the recombinant protein were identical to the amino acid sequence of native CMV PR, except for mutations introduced at the internal cleavage site to eliminate autoproteolysis at that site. The hexahistidinyl tag was placed downstream of amino acid 262 of the native CMV PR sequence. In this design, the Ala-Ser bond at amino acids 256-257 constitutes a site naturally cleaved by the protease during capsid maturation. The 268-amino-acid polypeptide with the (His)6 tag was expressed at high levels in E. coli as inclusion bodies. After solubilization of the inclusion bodies, the protease was purified to homogeneity by a single step using Ni2+ affinity chromatography. The protease was refolded to an active enzyme using dialysis which leads to effective autocleavage of the Ala-Ser bond at amino acids 256-257 to remove 12 amino acids including the (His)6 tag from the C terminus of the protein. This strategy yielded large amounts of highly purified CMV PR with the native N terminus and C terminus. Approximately 40 mg of purified CMV PR was obtained per liter of cell culture using this strategy. The enzymatic activity of CMV PR purified from inclusion bodies and refolded to an active enzyme was similar to the enzymatic activity of CMV PR expressed as a soluble protein in E. coli. In addition, the refolded CMV PR could be crystallized for X-ray diffraction.
Asunto(s)
Citomegalovirus/enzimología , Histidina , Serina Endopeptidasas/aislamiento & purificación , Secuencia de Aminoácidos , Bacteriófago lambda/genética , Catálisis , Cristalización , Citomegalovirus/genética , Escherichia coli/genética , Vectores Genéticos/genética , Humanos , Cuerpos de Inclusión/enzimología , Datos de Secuencia Molecular , Péptidos/química , Pliegue de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Alineación de Secuencia , Serina Endopeptidasas/biosíntesis , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Solubilidad , Especificidad por SustratoRESUMEN
An autolysis-resistant mutant of the HIV-I protease was employed for removal of metabolically stabilized and highly bioactive analogues of bovine growth-hormone-releasing factor (bGRF) from their larger either synthetic or recombinant precursors. The N-terminal four amino acids in two selected model GRF analogues, Y1IDAIFTSSYRKVLAQLSARKLLQDILSRQVF32-OH (I; GRF32) and Y1IDAIFTSSYRKVLAQLSARKLLQDILSRQ30-OH (IA; GRF30), conform well to the specificity of the HIV-I protease for residues in the P1' to P4' positions of its peptide substrates. A variety of amino acids were tried in the N-terminal extension (positions P4-P1) to fit the protease substrate specificity for the 8 amino acids in positions P4-P4'. A synthetic precursor of I, extended N-terminally with RQVF-, a sequence representing the four C-terminal residues in I, was effectively cleaved by the protease at the Phe-1-Tyr1 bond (... RQVF-decreases-YIDA ...) to release GRF32. However, when several soluble fusion proteins linked to GRF32 by the RQVF sequence were expressed in Escherichia coli, attempts to cleave out the core GRF32 met with variable, and only limited, success. By random mutagenesis in a propeptide segment, [MGQSVAQVF]-decreases-GRF30, (II) was identified as a construct that showed reasonably high-level expression in E. coli and was effectively processed by the HIV-I protease. A yield of 5 mg of pure GRF30 was obtained/litre of culture medium after a single HPLC purification step.
Asunto(s)
Hormona Liberadora de Hormona del Crecimiento/metabolismo , Proteasa del VIH/metabolismo , VIH-1/enzimología , Fragmentos de Péptidos/metabolismo , Precursores de Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Hormona Liberadora de Hormona del Crecimiento/síntesis química , Hormona Liberadora de Hormona del Crecimiento/genética , Modelos Químicos , Datos de Secuencia Molecular , Mutagénesis , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/genética , Precursores de Proteínas/síntesis química , Precursores de Proteínas/genética , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por SustratoRESUMEN
Active, recombinant p68 reverse transcriptase (RT) from human immunodeficiency virus type 2 (HIV-2), with an NH2-terminal extension containing a hexahistidine sequence was isolated from extracts of Escherichia coli by immobilized metal affinity chromatography. Treatment of the purified p68/p68 homodimer of HIV-2 RT with recombinant HIV-2 protease generates stable, active heterodimer (p68/p58) that is resistant to further hydrolysis. Analysis of this p68/p58 HIV-2 RT heterodimer revealed that while one subunit is intact p68, the p58 subunit is COOH-terminally truncated by cleavage, not at Phe440 as is seen in processing of the p66/p66 HIV-1 RT homodimer by HIV-1 protease, but at Met484. The expected COOH-terminal p10 fragment resulting from hydrolysis of p68 at Met484 is not released intact, but undergoes further cleavage at Asn494, Met503, and Tyr532. Processing of p68/p68 HIV-2 RT with the HIV-1 protease led to cleavage of the Phe440-Tyr441 bond, exactly as is seen with p66/p66 HIV-1 RT, to give the analogous p53 subunit. Studies of a peptide substrate modeled after residues 437-444 in HIV-2 RT showed that while the HIV-1 protease was able to cleave the Phe440 bond, this bond was resistant to cleavage by the HIV-2 enzyme. Our findings provide a rationale for the previous observation that the RT heterodimer isolated from HIV-2 lysates is larger than that from HIV-1. We conclude that the p68/p58 HIV-2 RT heterodimer, containing the Met484 truncated p58 subunit, is a biologically relevant form of the enzyme in vivo.
Asunto(s)
Proteasa del VIH/metabolismo , VIH-1/enzimología , VIH-2/enzimología , ADN Polimerasa Dirigida por ARN/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Transcriptasa Inversa del VIH , Hidrólisis , Datos de Secuencia Molecular , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , ADN Polimerasa Dirigida por ARN/genética , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Heparan sulfate proteoglycans at cell surfaces or in extracellular matrices bind diverse molecules, including growth factors and cytokines, and it is believed that the activities of these molecules may be regulated by the metabolism of heparan sulfate. In this study, purification of a heparan sulfate-degrading enzyme from human platelets led to the discovery that the enzymatic activity residues in at least two members of the platelet basic protein (PBP) family known as connective tissue activating peptide-III (CTAP-III) and neutrophil activating peptide-2. PBP and its N-truncated derivatives, CTAP-III and neutrophil activating peptide-2, are CXC chemokines, a group of molecules involved in inflammation and wound healing. SDS-polyacrylamide gel electrophoresis analysis of the purified heparanase resulted in a single broad band at 8-10 kDa, the known molecular weight of PBP and its truncated derivatives. Gel filtration chromatography of heparanase resulted in peaks of activity corresponding to monomers, dimers, and tetramers; these higher order aggregates are known to form among the chemokines. N-terminal sequence analysis of the same preparation indicated that only PBP and truncated derivatives were present, and commercial CTAP-III from three suppliers had heparanase activity. Antisera produced in animals immunized with a C-terminal synthetic peptide of PBP inhibited heparanase activity by 95%, compared with activity of the purified enzyme in the presence of the preimmune sera. The synthetic peptide also inhibited heparanase by 95% at 250 microM, compared to the 33% inhibition of heparanase activity by two other peptides. The enzyme was determined to be an endoglucosaminidase, and it degraded both heparin and heparan sulfate with optimal activity at pH 5.8. Chromatofocusing of the purified heparanase resulted in two protein peaks: an inactive peak at pI7.3, and an active peak at pI 4.8-5.1. Sequence analysis showed that the two peaks contained identical protein, suggesting that a post-translational modification activates the enzyme.
Asunto(s)
Factores de Coagulación Sanguínea/metabolismo , Plaquetas/metabolismo , Tejido Conectivo/metabolismo , Glucuronidasa , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Antagonistas de Heparina/sangre , Heparitina Sulfato/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Factores de Coagulación Sanguínea/química , Western Blotting , Células Cultivadas , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Embrión de Mamíferos , Glicosaminoglicanos/aislamiento & purificación , Glicosaminoglicanos/metabolismo , Glicósido Hidrolasas/aislamiento & purificación , Humanos , Focalización Isoeléctrica , Cinética , Pulmón , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Estructura Secundaria de Proteína , Proteoglicanos/aislamiento & purificación , Proteoglicanos/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Radioisótopos de Azufre , beta-TromboglobulinaRESUMEN
Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl ester from high- and low-density lipoproteins to triglyceride-rich lipoproteins, and reciprocally mediates triglyceride transfer. The gene for cynomolgus monkey CETP was expressed in serum-free CHO culture with 2 micrograms/ml insulin as its only exogenous protein supplement. Cell growth was facilitated by immobilizing the CHO cells in alginate beads. Recombinant CETP (rCETP) was purified 176-fold with a three-step protocol resulting in a 60% final yield as measured by a fluorescent CETP activity assay. Typically, 3.4 mg of rCETP was purified from 1700 ml of media by affinity-gel chromatography involving Reactive Red 120 (RR120) followed by concanavalin A Sepharose 4B and rechromatography on RR120. SDS-PAGE shows a single broad band of M(r) ranging from 68,000 to 74,000 which immunoreacts in Western blot analysis. Amino acid analysis and protein sequencing of the purified protein agree with the theoretical amino acid composition and sequence of cynomolgus CETP.
Asunto(s)
Proteínas Portadoras/biosíntesis , Proteínas Portadoras/aislamiento & purificación , Glicoproteínas , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Células CHO , Proteínas Portadoras/química , Proteínas Portadoras/genética , División Celular , Proteínas de Transferencia de Ésteres de Colesterol , Cromatografía de Afinidad , Clonación Molecular , Cricetinae , Medios de Cultivo , Expresión Génica/genética , Macaca fascicularis , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Análisis de SecuenciaRESUMEN
Site-directed mutagenesis of autolysis sites in the human immunodeficiency virus type 1 (HIV-1) protease was applied in an analysis of enzyme specificity; the protease served, therefore, as both enzyme and substrate in this study. Inspection of natural substrates of all retroviral proteases revealed the absence of beta-branched amino acids at the P1 site and of Lys anywhere from P2 through P2'. Accordingly, several mutants of the HIV-1 protease were engineered in which these excluded amino acids were substituted at their respective P positions at the three major sites of autolysis in the wild-type protease (Leu5-Trp6, Leu33-Glu34, and Leu63-Ile64), and the mutant enzymes were evaluated in terms of their resistance to autodegradation. All of the mutant HIV-1 proteases, expressed as inclusion bodies in Escherichia coli, were enzymatically active after refolding, and all showed greatly diminished rates of cleavage at the altered autolysis sites. Some, however, were not viable enzymatically because of poor physical characteristics. This was the case for mutants having Lys replacements of Glu residues at P2' and for another in which all three P1 leucines were replaced by Ile. However, one of the mutant proteases, Q7K/L33I/L63I, was highly resistant to autolysis, while retaining the physical properties, specificity, and susceptibility to inhibition of the wild-type enzyme. Q7K/L33I/L63I should find useful application as a stable surrogate of the HIV-1 protease. Overall, our results can be interpreted relative to a model in which the active HIV-1 protease dimer is in equilibrium with monomeric, disordered species which serve as the substrates for autolysis.
Asunto(s)
Proteasa del VIH/metabolismo , VIH-1/enzimología , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Proteasa del VIH/genética , Inhibidores de la Proteasa del VIH/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligopéptidos/metabolismo , Conformación Proteica , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The presence of Val2 in mGRF(1-42)OH is unique and, as shown in this study, renders this GRF resistant to plasma DPP-IV, the main enzyme responsible for rapid hydrolysis and inactivation of Ala2-containing GRFs from other species via cleavages between Ala2-Asp3. The presence of DPP-IV activity in mouse serum, and mouse and bovine plasma has been demonstrated with Gly-Pro-p-nitroanilide and/or with two DPP-IV-sensitive bGRF analogs, [Leu27]bGRF(1-29)NH2 and [Ala15,Leu27]bGRF(1-29)NH2, which were effectively converted to their respective (3-29) fragments. During incubations of mGRF(1-42)OH in mouse serum or plasma, as well as in bovine plasma in vitro, no major fragments were detectable, except for small amounts of metabolites with HPLC retention times corresponding to those of mGRF(12-42)OH and mGRF(21-42)OH, indicative of possible trypsin-like cleavages between Arg11-Lys12 and Arg20-Lys21. Both mGRF(1-42)OH (t1/2 52-78.5 min) and [Val2,Ala15,Leu27]-bGRF(1-29)NH2 (t1/2 78.5 min) disappeared 5 to 7 times faster in mouse than in bovine plasma, indicating much higher activity of various degrading enzymes in mouse plasma. In summary, our data provide evidence that mGRF(1-42)OH, despite its resistance to plasma DPP-IV, is degraded relatively fast in mouse plasma or serum because of trypsin-like and other, non-DPP-IV-related, proteolytic cleavages.
Asunto(s)
Sangre/metabolismo , Endopeptidasas/sangre , Hormona Liberadora de Hormona del Crecimiento/análogos & derivados , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Dipeptidil Peptidasa 4 , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/sangre , Ratones , Datos de Secuencia Molecular , Especificidad de la EspecieRESUMEN
We have previously identified hsp56, a protein component of steroid receptor complexes, as an FK506 binding protein [Yem et al. (1992) J. Biol. Chem. 267, 2868-2871]. We now report that hsp56 is also found to be a major immunophilin in chicken thymus, by virtue of binding to FK506-Affi-Gel-10 as well as positive cross-reactivity with a polyclonal antiserum directed against human hsp56. Limited digests of purified chicken hsp56 with endoproteinase Lys C result in the production of a unique polypeptide having a mass of about 17 kDa (p17), as judged by Western blotting. Peptide mapping provided additional proof that p17 is a fragment which comprises the entire FK506 binding domain I of chicken hsp56, terminating with an Arg-Lys which might represent a processing site. Binding of radiolabeled dihydro FK506 to p17 is saturable with a calculated KD of 42 nM. Since size exclusion chromatography of drug-p17 complexes indicates that the active species is a homodimer with a mass of 30-40 kDa, the stoichiometry calculated for the drug-protein complex is approximately 1:1. Furthermore, unlike FKBP-12, chicken p17 bound to FK506 does not bind to calcineurin-calmodulin complexes. This work demonstrates the excision of a domain from an hsp56 protein that is active in binding FK506 and functionally distinct from FKBP-12, a protein of similar size and structure.