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1.
J Thromb Haemost ; 4(7): 1559-65, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16839354

RESUMO

BACKGROUND: A single site mutant (M5) of prourokinase (proUK) was developed to make proUK less vulnerable to spontaneous activation in plasma. This was a problem that seriously compromised proUK in clinical trials, as it precluded proUK-mediated fibrinolysis at therapeutic concentrations. METHODS AND RESULTS: After completing dose-finding studies, 12 anesthetized dogs with femoral artery thrombosis were given either M5 (2.0 mg kg(-1)) or tissue plasminogen activator (t-PA) (1.4 mg kg(-1)) by i.v. infusion over 60 min (20% administered as a bolus). Two pairs of standardized injuries were inflicted at which hemostasis was completed prior to drug administration. Blood loss was quantified by measuring the hemoglobin in blood absorbed from these sites. Thrombolysis was evaluated at 90 min and was comparably effective by both activators. Rethrombosis developed in one t-PA dog. The principal difference found was that blood loss was 10-fold higher with t-PA (mean approximately 40 mL) than with M5 (mean approximately 4 mL) (P = 0.026) and occurred at more multiple sites (mean 2.7 vs. 1.2). This effect was postulated to be related to differences in the mechanism of plasminogen activation by t-PA and M5 in which the latter is promoted by degraded rather than intact (hemostatic) fibrin. In addition, two-chain M5 was efficiently inactivated by plasma C1 inactivator, an exceptional property which helped contain its non-specific proteolytic effect. CONCLUSIONS: Intravascular thrombolysis by M5 was accompanied by significantly less bleeding from hemostatic sites than by t-PA. This was attributed to the proUK paradigm of fibrinolysis being retained at therapeutic concentrations by the mutation.


Assuntos
Hemorragia/induzido quimicamente , Hemostasia/efeitos dos fármacos , Mutação , Terapia Trombolítica/métodos , Ativador de Plasminogênio Tecidual/administração & dosagem , Ativador de Plasminogênio Tipo Uroquinase/administração & dosagem , Ativador de Plasminogênio Tipo Uroquinase/genética , Animais , Modelos Animais de Doenças , Cães , Estabilidade Enzimática/genética , Artéria Femoral , Fibrinólise/efeitos dos fármacos , Fibrinólise/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Terapia Trombolítica/efeitos adversos , Trombose/tratamento farmacológico
2.
Nat Struct Biol ; 8(7): 611-5, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11427892

RESUMO

Certain chemokines act as natural antagonists of human immunodeficiency virus (HIV) by blocking key viral coreceptors, such as CCR5 and CXCR4, on the surface of susceptible cells. Elucidating the structural determinants of the receptor-binding and HIV-inhibitory functions of these chemokines is essential for the rational design of derivative molecules of therapeutic value. Here, we identify the structural determinants of CCR5 recognition and antiviral activity of the CC chemokine RANTES, showing that critical residues form a solvent-exposed hydrophobic patch on the surface of the molecule. Moreover, we demonstrate that the biological function is critically dependent on dimerization, resulting in the exposure of a large ( approximately 180 A2), continuous hydrophobic surface. Relevant to the development of novel therapeutic approaches, we designed a retroinverted RANTES peptide mimetic that maintained both HIV- and chemotaxis-antagonistic functions.


Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Quimiocina CCL5/química , Quimiocina CCL5/metabolismo , HIV-1/metabolismo , Receptores CCR5/metabolismo , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Fármacos Anti-HIV/farmacologia , Antagonistas dos Receptores CCR5 , Linhagem Celular , Quimiocina CCL5/análogos & derivados , Quimiocina CCL5/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Técnicas de Cocultura , Dimerização , Desenho de Fármacos , Células Gigantes/efeitos dos fármacos , Células Gigantes/virologia , HIV-1/efeitos dos fármacos , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Modelos Moleculares , Mimetismo Molecular , Mutagênese/genética , Peptídeos/síntese química , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/farmacologia , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Reprodutibilidade dos Testes , Relação Estrutura-Atividade
3.
Biochemistry ; 35(45): 14070-6, 1996 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-8916892

RESUMO

Single-chain urokinase-type plasminogen activator or pro-urokinase is a zymogen with an intrinsic catalytic activity which is greater than that of most other zymogens. To study the structural basis for this activity, a three-dimensional homology model was calculated using the crystallographic structure of chymotrypsinogen, and the structure-function relationship was studied using site-directed mutagenesis and kinetic analysis. This model revealed a unique Lys300 in pro-urokinase which could form a weak interaction with Asp355, adjacent to the active site Ser356. It was postulated that this lysine, by its epsilon-amino group, may serve to pull Ser356 close to the active position, thereby inducing the higher intrinsic activity of pro-urokinase. This was consistent with the published finding that a homologous lysine (Lys416) in single chain tissue plasminogen activator when mutated to serine induced some reduction in activity. To test this hypothesis, a site-directed mutant with a neutral residue (Lys300-->Ala) was produced and characterized. The Ala300-pro-urokinase had a 40-fold lower amidolytic activity than that of pro-urokinase. It was also stable in plasma at much higher concentrations than pro-urokinase, reflecting much attenuated plasminogen activation. Plasmin activatability was comparable to that of pro-urokinase, but the resultant two-chain derivative (Ala300-urokinase) had a lower enzymatic activity (approximately 33% that of urokinase) due to a reduction of kcat. Interestingly, the KM of two-chain Ala300-urokinase against plasminogen was 5.8-fold lower than that of urokinase, being similar to that of pro-urokinase which has a KM about 5-fold lower than urokinase. In conclusion, the hypothesis that Lys300 is a key structural determinant of the high intrinsic activity of pro-urokinase was confirmed by these studies. This residue also appears to be important for the full expression of the enzymatic activity of urokinase.


Assuntos
Precursores Enzimáticos/química , Serina Endopeptidases/química , Ativador de Plasminogênio Tipo Uroquinase/química , Ativação Enzimática , Fibrinólise , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Proteínas Recombinantes , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Especificidade por Substrato , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
4.
J Biotechnol ; 30(3): 367-78, 1993 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-7764112

RESUMO

We have expressed in Escherichia coli a soluble, truncated form of the human 55 kDa Tumor Necrosis Factor (TNF) receptor. For this purpose a plasmid was constructed which contains the extracellular domain of the 55 kDa TNF receptor fused to the coding sequence of the IgG binding domains of protein A from Staphylococcus aureus. The fusion product (TNFR-PA) obtained in E. coli is a soluble protein which bound human TNF alpha (huTNF alpha) with high affinity. In ligand-blotting experiments huTNF alpha bound to a single 52 kDa protein, a molecular mass corresponding to that expected for the monomeric fusion product. In gel filtration experiments binding activity was recovered from fractions that eluted at a volume corresponding to 140-150 kDa. TNFR-PA neutralized huTNF alpha in an in vitro cytotoxicity assay.


Assuntos
Escherichia coli/metabolismo , Receptores do Fator de Necrose Tumoral/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Cromatografia em Gel , Clonagem Molecular , Humanos , Peso Molecular , Receptores do Fator de Necrose Tumoral/genética , Solubilidade , Proteína Estafilocócica A/biossíntese
5.
Biochem Biophys Res Commun ; 194(2): 978-85, 1993 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-8343178

RESUMO

Single-chain urokinase-plasminogen activator contains 24 cysteine residues involved in 12 disulfide bonds and distributed all along the three domains of the protein. In order to investigate the role of these disulfide bridges in the catalytic activities of scu-PA, we used site-specific mutagenesis to construct 10 mutants in which some cysteine residues were changed to serine residues. Each mutated DNA fragment was cloned into a procaryotic expression vector and the protein expressed in E. coli. Mutant proteins of the expected size were produced and analyzed for amidolytic and fibrinolytic activities. From this, it is shown that: i) the disulfide bonds in the epidermal growth factor (EGF)-like and in the kringle domains are not necessary. Moreover, disulfide bond deletion in the kringle domain improves those catalytic activities; ii) on the contrary, the disulfide bridges in the catalytic domain are essential for maintaining both activities.


Assuntos
Cisteína , Mutagênese Sítio-Dirigida , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Dissulfetos/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Fibrina/metabolismo , Fibrinólise , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/isolamento & purificação
6.
Eur J Biochem ; 214(1): 295-304, 1993 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-7685281

RESUMO

Novel hirudin variants isolated from the leech Hirudinaria manillensis, a leech more specialized for mammalian parasitism, are described. Isolation of antithrombin polypeptides was performed by ion-exchange chromatographies followed by an affinity chromatography step on immobilized thrombin. The major active component, antithrombin polypeptide peak 2 (HM2) and a second polypeptide, named HM1, were purified to homogeneity and their complete amino acid sequences were determined. The protein structure of the two hirudin variants include 64 amino acids with 6 cysteine residues at highly conserved positions. Comparison of the amino acid sequences of HM1 and HM2 with other known hirudins shows differences mainly in the central part and in the C-terminal region of the polypeptides. Particularly significant is the lack of a sulfated tyrosine residue in the C-terminal portion of the molecule which is replaced by aspartic acid. Polymerase chain reaction cloning techniques were used to isolate and characterize the cDNAs and determine the genomic structures of these hirudin-like polypeptides. The cDNA clones coding for the two variants indicate the expression of pre-hirudins of 84 amino acids where the first 20 residues constitute the signal peptide required for extracellular secretion. The leader sequence appears to be highly conserved for both isoforms and shares a complete similarity with the partial hirudin variant 2 (HV2) signal peptide sequence previously reported. The HM1 and HM2 gene fragments show the presence of four exons: the first one corresponding to a 20-amino-acid signal peptide while the other three exons share the full primary structure of the antithrombin polypeptides. HM2 was also efficiently produced in recombinant Escherichia coli by expressing a periplasmic construction containing the synthetic gene.


Assuntos
Hirudinas/química , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Cromatografia de Afinidade , Cromatografia por Troca Iônica , DNA/química , DNA/genética , Escherichia coli/metabolismo , Hirudinas/genética , Hirudinas/isolamento & purificação , Hirudinas/farmacologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA/genética , Proteínas Recombinantes/biossíntese , Trombina/antagonistas & inibidores
7.
J Clin Invest ; 91(3): 1149-56, 1993 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8450044

RESUMO

Endothelin-1, initially identified as potent vasoconstrictor secreted by vascular endothelial cells, was subsequently found to have many effects on both vascular and nonvascular tissues. We have identified from a human placenta cDNA library a clone (cDNA-2) which corresponds to a novel 5'-extended preproendothelin 1 (preproET-1) mRNA. Comparison with the known preproET-1 mRNA (cDNA-1), showed that the two molecules share the same coding sequence but differ in the 5'-untranslated region. Interestingly, cDNA-2 extends upstream of promoter regions previously shown to be essential for full preproET-1 expression. Primer extension and PCR analysis of human placenta RNA demonstrated the presence of additional transcription initiation sites located upstream of the previously identified preproET-1 CAP site. Moreover, the two mRNAs show different pattern of expression. To elucidate the mechanisms controlling the production of alternative transcripts we transfected COS-1 cells with a series of preproET-1 promoter deletion mutants. This analysis revealed that the human preproET-1 gene can be transcribed from a proximal and a distal promoter element which has hitherto been undetected. In addition, we demonstrate the presence of a region in the down-epithelial specific expression.


Assuntos
Endotelinas/genética , Regiões Promotoras Genéticas , Precursores de Proteínas/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Clonagem Molecular , DNA/genética , DNA/isolamento & purificação , Endotelina-1 , Feminino , Biblioteca Gênica , Células HeLa , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Placenta/fisiologia , Gravidez , RNA Mensageiro/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Transfecção
8.
Growth Factors ; 9(1): 31-9, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-8347350

RESUMO

When granulocyte-macrophage colony-stimulating factor (GM-CSF) is chemically conjugated to the ribosome-inactivating protein saporin, the resulting protein conjugate is highly toxic for cells expressing the GM-CSF receptor. Structural and Western blot analyses of the purified conjugate establish that it contains equimolar amounts of the starting materials and is free of any contamination by the non-conjugated components. The resulting bifunctional reagent is specifically cytotoxic to cells expressing the GM-CSF receptor, but is ineffective to cells that do not express the receptor. The cytotoxic activity is inhibited in a dose-dependent manner by GM-CSF, but not by any one of five other peptide growth factors. This is the first report of a mitotoxin for cells that express the GM-CSF receptor and which promises to be a valuable tool to study the expression of the GM-CSF receptor in normal and pathological states.


Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Imunotoxinas , N-Glicosil Hidrolases , Proteínas de Plantas/toxicidade , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Reagentes de Ligações Cruzadas/administração & dosagem , Reagentes de Ligações Cruzadas/isolamento & purificação , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Camundongos , Mitógenos/administração & dosagem , Proteínas de Plantas/administração & dosagem , Proteínas Inativadoras de Ribossomos Tipo 1 , Ribossomos/efeitos dos fármacos , Saporinas
9.
Eur J Biochem ; 210(3): 823-29, 1992 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-1336458

RESUMO

The functional features of a recombinant fibroblast growth factor (FGF) receptor (FGF-R) were investigated by expressing at high level in Escherichia coli a soluble non-glycosylated form of FGF-R1. The extracellular domain of the mature protein (XC-FGF-R), comprising the first 356 amino acids, was purified from a large-scale fermentation. After cell lysis, the protein was quantitatively found in the pellet. XC-FGF-R was solubilized using guanidine/HCl and allowed to refold using two dialysis steps. The refolded protein was obtained in a homogeneous form after ammonium sulphate precipitation and gel-filtration chromatography. The soluble receptor had the ability to form a complex with recombinant human basic FGF (rhbFGF) in solution, as demonstrated by immunoprecipitation with anti-(FGF-R) serum. Formation of a rhbFGF/XC-FGF-R complex was visualized by cross-linking experiments. Quantitative binding experiments with the XC-FGF-R immobilized on Affi-Gel resin showed high binding affinity for 125I-bFGF (Kd = 5-10 nM). Purified XC-FGF-R inhibited binding of 125I-bFGF to its high-affinity receptors on baby hamster kidney cells. These data suggest that glycosylation of the FGF-R is not necessary for its ligand-binding activity. The use of an E. coli expression system resulted in the efficient production of a soluble receptor in a form suitable for ligand/receptor structural studies and screening of new potential agonists and antagonists of angiogenesis. These results indicate that E. coli can be used for the production of complex molecules such as Ig-like receptors.


Assuntos
Escherichia coli/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Receptores Proteína Tirosina Quinases , Receptores de Superfície Celular/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos , Animais , Sequência de Bases , Ligação Competitiva , Linhagem Celular , Clonagem Molecular , Glicosilação , Humanos , Cinética , Dados de Sequência Molecular , Peso Molecular , Oligodesoxirribonucleotídeos , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
10.
Am J Pathol ; 141(3): 661-71, 1992 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-1325742

RESUMO

Lesions excised from nine patients undergoing surgery for Dupuytren's contracture (DC) and three normal fascia were examined for the presence of the angiogenic protein basic fibroblast growth factor (basic FGF). Endothelial cell proliferation assays established basic FGF-like activity in extracts of DC. Western blotting confirmed the presence of an 18,000-dalton protein which was localized in the lesions by immunohistochemical staining. All of the cells implicated in the progression of the disease (endothelial cells, fibroblasts, and myofibroblasts) contain the growth factor. Endothelial cells within the narrowed or occluded vessels, as well as fibroblasts surrounding these vessels, stained intensely positive. In situ hybridization using an antisense probe for human basic FGF and its receptor's (FGFR-1) mRNA established the major difference between normal and DC tissues: their levels are significantly higher than in the normal tissues. Thus the cells in DC also express both basic FGF and FGFR-1, suggesting a potential autocrine/paracrine role for basic FGF in the pathogenesis of DC. This finding is thus the first description of a nontumoral proliferative disease that can be directly associated with increased basic FGF mRNA. The possibility that therapies can be developed on the basis that basic FGF and its receptor are expressed in DC is discussed.


Assuntos
Contratura de Dupuytren/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Contratura de Dupuytren/patologia , Endotélio/citologia , Substâncias de Crescimento/metabolismo , Humanos , Imuno-Histoquímica , Hibridização de Ácido Nucleico , Receptores de Superfície Celular/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos
11.
Biochem Biophys Res Commun ; 186(2): 753-9, 1992 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-1497664

RESUMO

To investigate biochemical and biological parameters involved in preproendothelin-1 (preproET-1) maturation we infected Spodoptera frugiperda (Sf21) cells with a suitable engineered baculovirus vector carrying the cDNA encoding the entire human 212 amino acids precursor. Culture supernatants were tested by RIA using an anti-ET-1 serum, ET-1-like immunoreactive material (IRM) was detected in the infected Sf21 cells but not in control, wild-type or mock-infected cells. Fractionation of the culture supernatant by RP-HPLC coupled to an ET-1 specific RIA yielded two main peaks corresponding to the retention times of human bigET-1 and ET-1. Furthermore, culture supernatant of preproET-1 expressing Sf21 cells elicited a characteristic dose-response vasoconstrictive activity on rabbit vena cava, consistent with the amount of ET-1 as estimated by RP-HPLC coupled to RIA. These results suggest that insect cells possess the enzymatic activities necessary for human preproET-1 full maturation even though no such peptide has ever been found in insect cells.


Assuntos
Baculoviridae/genética , Endotelinas/metabolismo , Vetores Genéticos , Precursores de Proteínas/metabolismo , Animais , Sequência de Bases , Bioensaio , Linhagem Celular , Endotelina-1 , Endotelinas/genética , Endotelinas/farmacologia , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Mariposas , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Oligodesoxirribonucleotídeos , Precursores de Proteínas/genética , Coelhos , Transfecção , Vasoconstrição/efeitos dos fármacos , Veias Cavas/efeitos dos fármacos , Veias Cavas/fisiologia
12.
Biochem Biophys Res Commun ; 185(3): 1098-107, 1992 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-1378264

RESUMO

Basic residues Arg-118, Lys-119, Lys-128, and Arg-129 within a putative heparin-binding and receptor-binding region of the 155-amino acid form of basic fibroblast growth factor (bFGF) have been changed to neutral glutamine residues by site-directed mutagenesis of the human bFGF cDNA. The bFGF mutant (M6B-bFGF) was expressed in E. coli and purified to homogeneity. When compared to wild type bFGF, M6B-bFGF showed in cultured endothelial cells a similar receptor-binding capacity and mitogenic activity, but a reduced affinity for heparin-like low affinity binding sites, a reduced chemotactic activity, and a reduced capacity to induce the production of urokinase-type plasminogen activator. In vivo, M6B-bFGF lacked a significant angiogenic activity. Modifications of both the primary and the tertiary structure of bFGF appear to be responsible for the modified biological properties of M6B-bFGF, thus confirming the possibility to dissociate at the structural level some of the biological activities exerted by bFGF on endothelial cells.


Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Heparina/metabolismo , Mutagênese Sítio-Dirigida , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Bovinos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Membrana Celular/metabolismo , Quimiotaxia/efeitos dos fármacos , Clonagem Molecular , Endotélio Vascular , Indução Enzimática , Fator 2 de Crescimento de Fibroblastos/genética , Humanos , Cinética , Dados de Sequência Molecular , Neovascularização Patológica , Oligodesoxirribonucleotídeos , Plasmídeos , Ativadores de Plasminogênio/biossíntese , Receptores de Fatores de Crescimento de Fibroblastos
14.
J Biotechnol ; 21(1-2): 83-92, 1991 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-1367692

RESUMO

The 146-amino acid form of basic fibroblast growth factor (bFGF) was expressed in Escherichia coli and purified by a two step process including ion exchange and heparin-Sepharose chromatographies. However, the resulting protein consisted of a mixture of 146- and 145-amino acid forms, indicating that, besides the initial methionine, also the following residue (proline) was removed from the N-terminus. The same phenomenon was observed when the 155-amino acid form, which is biologically equivalent to the shorter one, was expressed in E. coli. Taking into account the previously known data concerning the possible mechanism of cleavage of the extended forms of bFGF in vivo, we developed an efficient enzymatic process that allows the production of an homogeneous 146-amino acid form from recombinant NH2-end extended forms. This process takes advantage of the protecting effect that heparin exerts on bFGF. Accordingly, when bFGF, complexed to heparin, is treated with pepsin A, an aspartic protease with a broad specificity, only the Leu9-Pro10 peptide bond is cleaved generating the 146-amino acid form. Quantitative yields of this reaction are also achieved when bFGF is bound to a heparin-Sepharose column, allowing the integration of this enzymatic step directly during purification of the recombinant extended forms of bFGF.


Assuntos
Fator 2 de Crescimento de Fibroblastos/biossíntese , Sequência de Aminoácidos , Bioensaio , Cromatografia de Afinidade , Fator 2 de Crescimento de Fibroblastos/farmacologia , Hidrólise , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia
15.
J Biotechnol ; 20(2): 201-12, 1991 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-1367547

RESUMO

Urokinase-type plasminogen activator (uPA) is a proteolytic enzyme able to convert the zymogen plasminogen into the strong protease plasmin. The availability of very sensitive tests to measure the enzymatic activity of a plasminogen activator renders the corresponding gene an ideal candidate for the detection of promoter activity. In this paper we describe the utilization of the human uPA gene as detector of tissue-specificity of the murine whey acidic protein (WAP) expression signals in transgenic mice. The WAP promoter has been previously investigated for the production of foreign proteins in the milk of transgenic animals. In our genetic constructions, the human uPA cDNA was linked to the promoter region as well as to 3'-end distal sequences of the WAP gene. Five transgenic lines were obtained in which, however, expression levels of human uPA in the milk were still quite low. Surprisingly, four of these five positive transgenic mice show a consistent activity of the WAP promoter in brain extracts compared to other tissues.


Assuntos
Proteínas do Leite/genética , Ativadores de Plasminogênio/genética , Ativador de Plasminogênio Tipo Uroquinase/genética , Animais , Química Encefálica , Clonagem Molecular , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Leite/química , Proteínas do Leite/análise , Especificidade de Órgãos , Ativadores de Plasminogênio/análise , Regiões Promotoras Genéticas , Ativador de Plasminogênio Tipo Uroquinase/análise
16.
Gene ; 101(2): 255-60, 1991 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-1647362

RESUMO

The thrombin inhibitor, hirudin, from the leech Hirudo Medicinalis, is the most powerful natural anticoagulant known. It has been characterized as a polypeptide of 65 amino acids which exhibits its anticoagulant properties by binding tightly and specifically to alpha-thrombin. The potency and specificity of hirudin have generated interest on its possible use in the treatment or prophylaxis of various thrombotic diseases. We have used the baculovirus expression system to efficiently produce active hirudins in insect cells. The Autographa californica nuclear polyhedrosis virus has proved useful as a helper-independent viral expression vector for high-level production of recombinant proteins in cultured insect cells. Hirudin variants (HV1 and HV2) were produced in infected insect cells as secreted proteins by joining their coding sequences to the leader peptide sequence of the vescicular stomatitis virus G protein. The recombinant products were biologically active and, interestingly, N-terminal sequencing of HV1 revealed that the heterologous leader peptide is correctly removed.


Assuntos
Baculoviridae/genética , Hirudinas/biossíntese , Glicoproteínas de Membrana , Proteínas Recombinantes/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Hirudinas/genética , Hirudinas/metabolismo , Cinética , Sanguessugas/genética , Dados de Sequência Molecular , Mariposas , Sinais Direcionadores de Proteínas , Vírus da Estomatite Vesicular Indiana/genética , Proteínas do Envelope Viral/genética
17.
Proc Natl Acad Sci U S A ; 88(7): 2628-32, 1991 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-1849269

RESUMO

A recombinant deletion mutant of the 155-amino acid form of human basic fibroblast growth factor (bFGF), lacking amino acid residues 27-32 (Lys-Asp-Pro-Lys-Arg-Leu), was expressed in Escherichia coli and purified to homogeneity by heparin-Sepharose affinity chromatography. When maintained in the presence of an equimolar concentration of soluble heparin, the bFGF mutant (M1-bFGF) is as potent as bFGF in stimulating cell proliferation in normal and transformed fetal bovine aortic endothelial cells, in adult bovine aortic endothelial cells, and in NIH 3T3 fibroblasts. However, under the same experimental conditions, M1-bFGF is at least 100 times less efficient than bFGF in stimulating plasminogen activator (PA) production in endothelial cells, as assayed by chromogenic PA assay, SDS/PAGE zymography, and Northern blot analysis of urokinase-type PA mRNA. In the presence of heparin, M1-bFGF binds to bFGF plasma membrane receptors present on endothelial cells in a manner undistinguishable from bFGF. It also induces the same tyrosine phosphorylation pattern when added to NIH 3T3 cells. The data suggest that the PA-inducing activity of bFGF may depend upon a functional domain that differs from those involved in the mitogenic activity of the growth factor and that the binding of bFGF to its plasma membrane receptor may not be sufficient to induce urokinase-type PA production in endothelial cells.


Assuntos
Fator 2 de Crescimento de Fibroblastos/genética , Mitógenos , Ativadores de Plasminogênio , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Deleção Cromossômica , Replicação do DNA/efeitos dos fármacos , Regulação para Baixo , Fator 2 de Crescimento de Fibroblastos/isolamento & purificação , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Genes Sintéticos , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Sondas de Oligonucleotídeos , Plasmídeos , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos
18.
Blood Coagul Fibrinolysis ; 2(1): 113-20, 1991 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-1772978

RESUMO

Recombinant DNA technologies now allow the preparation of virtually any polypeptide sequence. Very efficient expression systems for prokaryotic and eukaryotic cells have been developed which may yield large quantities of the desired protein. Bacterial systems are still the most widely used while alternative organisms are often considered when post-translational modifications could influence the biological behaviour of the product. For hirudin or its analogues, two important molecular characteristics should be taken into account. First, it is necessary that no extra amino acid residue, such as the initial methionine, is present on the NH2 end of the recombinant polypeptide. It is known that a free N-terminal sequence is crucial for the thrombin inhibitory activity. Second, a sulphate group on tyrosine at position 63 is found in natural hirudin extracted from leeches. Such post-translational modification has never been observed for all the recombinant hirudin preparations reported to date even though the importance of the sulphate group on the in vitro and in vivo activity of hirudin has not yet been clarified. Finally, the recombinant DNA methodology of choice for the commercial development of hirudin must also take into consideration yield and cost factors which ultimately will affect the widespread use of this product particularly if it has to compete with heparin. We will review our work on the preparation of recombinant hirudin describing bacterial and insect cell expression systems and addressing some of the questions mentioned above.


Assuntos
Hirudinas/análogos & derivados , Hirudinas/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/genética , Baculoviridae/genética , Sequência de Bases , Células Cultivadas , Clonagem Molecular/métodos , Escherichia coli/genética , Genes Sintéticos , Vetores Genéticos , Hirudinas/genética , Hirudinas/isolamento & purificação , Dados de Sequência Molecular , Mariposas , Fosforilação , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes , Tirosina/metabolismo
19.
Eur J Biochem ; 195(3): 691-7, 1991 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-1847867

RESUMO

Prourokinase is a plasminogen activator of 411 amino acids which displays a clot-lysis activity through a fibrin-dependent mechanism, and which seems to be a promising agent for the treatment of acute myocardial infarction. The preparation of recombinant prourokinase in bacteria has been hampered by its insolubility and by difficulty in refolding the polypeptide chain. In this paper we describe the renaturation process of two recombinant proteins expressed in Escherichia coli as inclusion bodies: prourokinase and a deletion derivative (delta 125-prourokinase) in which 125 amino acids of the N-terminal region have been removed. Deletion of this sequence brings to higher refolding yields and faster kinetics (first-order rate constant of renaturation of 0.57 h-1 for delta 125-prourokinase and 0.25 h-1 for prourokinase). Our process involves sequential steps of denaturation, reduction and controlled refolding of the polypeptide chain. When applied to pure, non-glycosylated and active prourokinase, it gives a refolding yield of about 80%, demonstrating the efficiency of the renaturation procedure. Lower yields (15% and 30%, respectively, for prourokinase and delta 125-prourokinase) were obtained when the same refolding protocol was applied to inclusion bodies from bacteria. After purification to homogeneity (as shown by HPLC and SDS/PAGE) specific activities were 160,000 and 250,000 IU/mg protein, respectively, for prourokinase and delta 125-prourokinase. As with prourokinase, the deletion mutant delta 125-prourokinase displays a zymogenic nature, being activated by plasmin to the active two-chain form; however, this mutant is approximately fourfold more resistant than prourokinase to plasmin activation, and consequently shows a different fibrinolytic profile.


Assuntos
Escherichia coli/genética , Fibrinolíticos/metabolismo , Corpos de Inclusão/enzimologia , Ativadores de Plasminogênio/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cromatografia , Cromatografia em Gel , Deleção Cromossômica , Durapatita , Escherichia coli/enzimologia , Humanos , Hidroxiapatitas , Cinética , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Plasmídeos , Ativadores de Plasminogênio/genética , Ativadores de Plasminogênio/isolamento & purificação , Ativadores de Plasminogênio/farmacologia , Conformação Proteica , Desnaturação Proteica , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Mapeamento por Restrição , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/isolamento & purificação , Ativador de Plasminogênio Tipo Uroquinase/farmacologia
20.
Ann N Y Acad Sci ; 638: 329-40, 1991.
Artigo em Inglês | MEDLINE | ID: mdl-1785810

RESUMO

Basic fibroblast growth factor is a polypeptide belonging to a family of natural proteins also known as heparin-binding growth factors endowed with a pleiotropism of biological activities, the most striking of which are related to wound healing. Large quantities of recombinant human basic fibroblast growth factor (rh-bFGF) of a clinical grade were obtained and used to undertake preclinical and clinical studies. In vivo the wound healing effect of rh-bFGF was evaluated in experimental targets such as the cornea and the tympanic membrane, showing a significantly increased epithelial healing rate in drug-treated animals. The deposition of labeled rh-bFGF after topical applications in ocular wounding models did not result in a systemic absorption of the intact rh-bFGF molecule. The acute and the subchronic toxicity studies undertaken after iv and topical administration of a stable pharmaceutical formulation of rh-bFGF did not result in irritation, and no signs of general toxicity were observed. Altogether these data permitted us to start recently with human studies, which are still ongoing, aimed to evaluate the tolerability and the activity of rh-bFGF on tegumental targets such as the cornea and the skin.


Assuntos
Fator 2 de Crescimento de Fibroblastos/uso terapêutico , Cicatrização , Animais , Clonagem Molecular , Doenças da Córnea/tratamento farmacológico , Fator 2 de Crescimento de Fibroblastos/administração & dosagem , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/farmacocinética , Humanos , Técnicas In Vitro , Coelhos , Ratos , Proteínas Recombinantes/uso terapêutico , Membrana Timpânica
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