RESUMO
Reteplase (recombinant plasminogen activator, rPA) is a mutant non-glycosylated tissue-type plasminogen activator (tPA) containing 355 amino acids with longer half-life and promising thrombolytic activity than its original counterpart, full length tPA. In this study, we aimed to produce and optimize the purification process of recombinant tissue-type plasminogen activator (tPA) known as Reteplase (rPA). Reteplase cDNA synthesized from total mRNA isolated from human placenta was PCR amplified, cloned into a pET-28a(+) E. coli expression vector and expressed in Rosetta-gami 2 E. coli (Novagenâ) host. rPA was expressed as an inclusion body in E. coli and its biological activity was achieved after single step solubilization, purification and refolding. We exploited the strategy of Slow Refolding using Gradual Dialysis (SRGD) in which a refolding buffer containing glutathione oxidized (1 mM GSSG) and glutathione reduced (3 mM GSH) and pH 9.0 was used. Using the SRGD method, we were able to successfully obtain the protein in its active form. We obtained 4.26 mg of active refolded protein from a 50 mL culture that was scaled up in a bioreactor. The purity and homogeneity of rPA was evaluated by SDS-PAGE, Western blotting and mass spectrometry. Circular dichroism spectroscopy was conducted to evaluate the refolding and stability of the refolded rPA in comparison to reference standard rPA. The thrombolytic potential of rPA was assessed by fibrin plate assay and In Vitro clot lysis assay. The presented protocol offers a viable approach for enhancing both the yield and refolding efficiency of reteplase, potentially resulting in an increase in yield.
Assuntos
Escherichia coli , Redobramento de Proteína , Proteínas Recombinantes , Ativador de Plasminogênio Tecidual , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/química , Ativador de Plasminogênio Tecidual/isolamento & purificação , Ativador de Plasminogênio Tecidual/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/biossíntese , Humanos , Expressão Gênica , Fibrinolíticos/química , Fibrinolíticos/isolamento & purificação , Clonagem MolecularRESUMO
Tissue-type plasminogen activator (tPA) is a serine protease that plays a crucial role in the fibrinolytic system. We increased the activity of tPA by splicing the active site of dodder-cuscutain gene to human tPA. The chimeric cDNA of tPA was constructed by Splicing by Overlap Extension Polymerase Chain Reaction (SOEing-PCR) method and transferred to the hairy roots of tobacco using different strains of Agrobacterium rhizogenes. Chimeric-tPA was purified by lysine-sepharose chromatography and specific aptamers were designed using SELEX method. Multi wall carbon nanotubes were functionalized with selected aptamers, packed in a column, and used for purification. The results demonstrated that selected aptamer having KD values of 0.320 nM and IC50 of 28.9 nM possessed good affinity to tPA, and the chimeric-tPA was properly purified by aptamer-chromatography. Hairy roots expressing chimeric-tPA and normal-tPA produced 900 and 450 ngmg-1 of total protein, respectively. The activities of chimeric-tPA and normal-tPA were 90 and 60 IUml-1, respectively. Compared to the normal-tPA, chimeric-tPA showed more activity.
Assuntos
Aptâmeros de Nucleotídeos/química , Cromatografia de Afinidade/métodos , Cuscuta/genética , Nicotiana/genética , Proteínas de Plantas/isolamento & purificação , Ativador de Plasminogênio Tecidual/isolamento & purificação , Agrobacterium/genética , Agrobacterium/metabolismo , Aptâmeros de Nucleotídeos/síntese química , Clonagem Molecular , Cuscuta/metabolismo , Ensaios Enzimáticos , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Nanotubos de Carbono/química , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Ligação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Técnica de Seleção de Aptâmeros , Ativador de Plasminogênio Tecidual/biossíntese , Ativador de Plasminogênio Tecidual/genética , Nicotiana/metabolismoRESUMO
Streptokinase is a valuable fibrinolytic agent used to cope with myocardial infarction and brain stroke. Despite its high efficiency in dissolving blood clots, streptokinase (SK) has no specificity in binding fibrin, causing some problems such as internal bleedings following its administration. To make streptokinase fibrin specific and limit the fibrinolytic process to the clot location, we engineered a chimeric streptokinase by fusing the fibrin binding Kringle 2 domain of tissue plasminogen activator (TPA) to the streptokinase N-terminal end. The chimeric SK construct (KSK) with inserted Kringle 2 domain was cloned into pET28a expression vector. The expression of recombinant protein was carried out in Escherichia coli origami (DE3) and confirmed by SDS-PAGE and Western blotting analyses. We used the chromogenic substrate S-2251 method to assess the specific activities of the chimeric and control wild-type proteins. Then, the two proteins were added in amounts with equal activity to fibrin clots of identical size. Finally, the supernatant above the fibrin clots was collected and subjected to the chromogenic assay to analyze the specificity of the chimeric protein. The specific activities of the chimeric and wild-type proteins were found to be 0.06 U/mg and 0.07 U/mg, respectively. Because of the binding of the chimeric protein to fibrin, the mean specific activity was significantly lower in the KSK supernatant (0.01) compared with the control (approximately 0.06) (p < 0.05). Our in vitro results indicate that the chimeric streptokinase protein has strong fibrin-specific activity compared to the wild-type protein. However, further in vivo studies are needed to evaluate its potential fibrinolytic effects.
Assuntos
Proteínas de Bactérias , Engenharia de Proteínas , Streptococcus/genética , Estreptoquinase , Ativador de Plasminogênio Tecidual , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Fibrina/química , Fibrina/metabolismo , Fibrinólise , Humanos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Streptococcus/enzimologia , Estreptoquinase/biossíntese , Estreptoquinase/química , Estreptoquinase/genética , Estreptoquinase/isolamento & purificação , Ativador de Plasminogênio Tecidual/biossíntese , Ativador de Plasminogênio Tecidual/química , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/isolamento & purificaçãoRESUMO
Plants are low cost platforms for the production of recombinant proteins, but their complexity renders the purification of plant recombinant proteins more difficult than proteins expressed in yeast or bacteria. Plastid transformation enables high-level expression of foreign genes and the accumulation of recombinant proteins in plastid organelles. Histidine (His) tags are widely used for affinity purification of recombinant proteins in a nickel column. The human tissue-type plasminogen activator (tPA) is one of the most important pharmaceutical recombinant proteins involved in the breakdown of blood clots in different parts of the body. The truncated form of the tissue plasminogen activator (K2S) has a longer plasma half-life, better diffusion into the clot, and higher fibrinolytic activity. In a construct designed to insert the K2S gene in the tobacco chloroplast, the sequence of six histidines and a factor Xa protease site was fused to the C-terminus of the K2S protein. The presence and amount of tPA recombinant protein in transplastomic tobacco plants was estimated by ELISA analysis using a specific antibody. The protein was purified from total soluble protein, insoluble protein aggregates and the protein was extracted from the isolated chloroplast using nickel resin and a chromatography column. After digestion of the purified protein with factor Xa, the presence of the purified tPA protein was confirmed by western blot analysis.
Assuntos
Nicotiana/genética , Proteínas Recombinantes/isolamento & purificação , Ativador de Plasminogênio Tecidual/isolamento & purificação , Fracionamento Químico , Cloroplastos/genética , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/metabolismo , Nicotiana/metabolismoRESUMO
BACKGROUND: Human tissue plasminogen activator (tPA) belongs to the serine protease family. It converts plasminogen into plasmin and is used clinically to treat thrombosis. Human tPA is composed of 527 amino acids residues and contains 17 disulfide bonds. Escherichia coli has been used only rarely for the efficient production of recombinant tPA. However, the functional expression of full-length tPA that contains multiple disulfide bonds on an industrial scale remains challenging. Here, we describe the soluble expression and characterization of full-length tPA by auto-induction in E. coli. RESULTS: We achieved optimal levels of gene expression, minimized negative effects related to the production of heterologous proteins, and optimized cytoplasmic yields. Three different E. coli strains, BL21 (DE3), Rosetta, and Origami 2, could express tPA using an auto-induction mechanism. In addition, similar yields of recombinant protein were produced at temperatures of 33, 35, and 37°C. The E. coli strain origami 2 could increase disulfide bond formation in cytoplasmic tPA and produce purified soluble recombinant protein (~0.9 mg/l medium). The full-length tPA was monomeric in solution, and fibrin plate assays confirmed that the recombinant tPA displayed serine protease activity. CONCLUSIONS: This is the first report that describes the heterologous expression of correctly folded active full-length tPA. This could provide valuable information for using prokaryotic auto-induction expression systems to produce tPA at industrial and pharmaceutical levels without in vitro refolding during the production step.
Assuntos
Escherichia coli/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ativador de Plasminogênio Tecidual/isolamento & purificação , Ativador de Plasminogênio Tecidual/metabolismo , Fibrinólise , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Solubilidade , Ativador de Plasminogênio Tecidual/química , Ativador de Plasminogênio Tecidual/genéticaRESUMO
In this study, a strategy of the construction of leaky strains for the extracellular production of target proteins was exploited, in which the genes mrcA, mrcB, pal and lpp (as a control) from Escherichia coli were knocked out by using single- and/or double-gene deletion methods. Then the recombinant strains for the expression of exogenous target proteins including Trx-hPTH (human parathyroid hormone 1-84 coupled with thioredoxin as a fusion partner) and reteplase were reconstructed to test the secretory efficiency of the leaky strains. Finally, the fermentation experiments of the target proteins from these recombinant leaky strains were carried out in basic media (Modified R media) and complex media (Terrific Broth media) in flasks or fermenters. The results demonstrated that the resultant leaky strains were genetically stable and had a similar growth profile in the complex media as compared with the original strain, and the secretory levels of target proteins into Modified R media from the strains with double-gene deletion (up to 88.9%/mrcA lpp-pth) are higher than the excretory levels from the strains with single-gene deletion (up to 71.1%/lpp-pth) and the host E. coliâ JM109 (DE3) (near zero). The highest level of extracellular production of Trx-hPTH in fermenters is up to 680 mg l(-1).
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica , Proteínas Recombinantes/metabolismo , Meios de Cultura/química , Escherichia coli/crescimento & desenvolvimento , Deleção de Genes , Instabilidade Genômica , Hormônio Paratireóideo/genética , Hormônio Paratireóideo/isolamento & purificação , Hormônio Paratireóideo/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Tiorredoxinas/genética , Tiorredoxinas/isolamento & purificação , Tiorredoxinas/metabolismo , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/isolamento & purificação , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
Recombinant tissue-type plasminogen activator (rt-PA) has been produced in different hosts. In this research, transgenic tobacco was selected for production of human rt-PA. Transgenic plants were analyzed by polymerase chain reaction (PCR) and reverse-transcription (RT)-PCR. The protein was extracted by Lysine Sepharose chromatography column and was further purified by HiTrap desalting column. The function of eluted protein was analyzed on zymography gel. The results showed that the 1.7-kb cDNA of tissue-type plasminogen activator (t-PA) (as well as a shortened 650-bp transcript of t-PA) has been expressed in transgenic plants. The anticipated 63-kD protein band and an additional 53-kD protein were observed in transgenic plants. Finally, zymography assay revealed that the purified rt-PA has anticipated appropriate activity comparable to a positive control drug (Alteplase). On the whole, we can say that transgenic tobacco is a good alternative host for production of t-PA.
Assuntos
Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Ativador de Plasminogênio Tecidual/biossíntese , Ativador de Plasminogênio Tecidual/isolamento & purificação , Agrobacterium tumefaciens , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Fibrinolisina/metabolismo , Humanos , Cinética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plasminogênio/metabolismo , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Ativador de Plasminogênio Tecidual/genética , Nicotiana/genética , Nicotiana/metabolismoAssuntos
Contaminação de Medicamentos , Fibrinolisina/farmacologia , Neurônios/efeitos dos fármacos , Ativador de Plasminogênio Tecidual/farmacologia , Animais , Células Cultivadas , Diálise , Fibrinolisina/isolamento & purificação , Camundongos , Peso Molecular , Ativador de Plasminogênio Tecidual/isolamento & purificaçãoRESUMO
Combined docking and molecular dynamics (MD) simulations are carried out for the rational design of affinity peptide ligand of tissue-type plasminogen activator (t-PA). Ten amino acids that have high affinity to three different regions of t-PA are identified by the amino acids location method on the basis of candidate pocket structure of t-PA. Then, 14 tetrapeptides are built and docked into the candidate pocket of t-PA. The absolute value of the D(score) calculated from the docking simulation is used to assess the affinity of a peptide for t-PA. Consequently, six tetrapeptides that have high D(score) values are selected and linked to a spacer arm of [NH(CH(2))(6)NH(2)] that is present on EAH Sepharose gel. The linked compounds are further evaluated by docking into the candidate pocket of t-PA. As a result, the tetrapeptide QDES with the highest D(score) value is selected. Molecular surface analysis with the MOLCAD program reveals that electrostatic interactions and hydrogen bonds (H-bonds) contribute to the affinity interactions between the tetrapeptide and t-PA. MD simulations indicate that QDES-t-PA complex keeps stable, and the distances between the carboxyl groups of Asp189, Gln192 and Asp194 and the charged amino group of glutamine change little. Moreover, all the nine H-bonds found in the docking simulation are confirmed by the MD simulations. It is also found that three water molecules act as bridges between the ligand and the protein pocket by hydrogen bonding. Finally, high binding affinity and specificity of the peptide ligand are confirmed by the purification of t-PA from crude porcine heart extract using the immobilized-ligand column for affinity chromatography.
Assuntos
Cromatografia de Afinidade/métodos , Desenho de Fármacos , Oligopeptídeos/síntese química , Ativador de Plasminogênio Tecidual/isolamento & purificação , Animais , Simulação por Computador , Ligação de Hidrogênio , Modelos Moleculares , Miocárdio/enzimologia , Suínos , Ativador de Plasminogênio Tecidual/químicaAssuntos
Transtornos Mentais/induzido quimicamente , Transtornos Relacionados ao Uso de Substâncias/complicações , Animais , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Metanfetamina/efeitos adversos , Camundongos , Morfina/efeitos adversos , Receptores Dopaminérgicos/fisiologia , Ativador de Plasminogênio Tecidual/isolamento & purificação , Ativador de Plasminogênio Tecidual/fisiologiaRESUMO
Dyes-based biomimetic affinity chromatography has been used to purify therapeutically useful proteins. In order to design novel biomimetic affinity ligands for purification of tissue-type plasminogen activator (t-PA), small molecular fragments were achieved to fit in S3/4 binding site of t-PA by structure-based ligand design method (InsightII/Ludi). Three biomimetic affinity ligands A, B, and C were then designed, synthesized, and proved to bind the target protein (t-PA), exceeding the binding capacity of the commercial p-amino benzamidine affinity matrix. The designed affinity matrix A showed high efficiency to purify sc-tpa from the crude samples with 18-fold of purification.
Assuntos
Benzamidinas/química , Materiais Biomiméticos/química , Cromatografia de Afinidade/métodos , Ativador de Plasminogênio Tecidual/isolamento & purificação , Triazinas/química , Benzamidinas/síntese química , Materiais Biomiméticos/síntese química , Humanos , Ligantes , Ativador de Plasminogênio Tecidual/química , Triazinas/síntese químicaRESUMO
Tissue plasminogen activator (tPA) was produced in ornithine transcarbamoylase (OTC) cells by introducing the tPA gene into OTC cells. OTC cells were originally derived from Chinese hamster ovary (CHO) cells and express the first two enzymes of the urea cycle, carbamoyl phosphate synthetase I (CPS I) and OTC. To investigate glycosylation variants, tPA variants produced in serum-supplemented culture medium of OTC-tPA cells were separated by lysine-Sepharose 4B chromatography. Unlike in previous studies that used lysine-Sepharose chromatography, two peaks were identified to correspond to eluted glycosylation variants type I and II and type II and the percentages of the type I and type II variants were found to be 23% and 77%, respectively. The biological activities of the type I and II and type II variants were twofold that of the Third International tPA Standard (98/714) produced in the CHO cell line, and the activity of type II variant was 12.6% higher than that of the type I and II variants. These results demonstrate that tPA produced in urea-cycle-enzyme-producing OTC cells have a very high biological activity and the percentage of type II variant which is very valuable for the biopharmaceutical industry is higher than that of any report using CHO cells.
Assuntos
Ornitina Carbamoiltransferase/fisiologia , Proteínas Recombinantes/biossíntese , Ativador de Plasminogênio Tecidual/biossíntese , Animais , Células CHO , Cromatografia , Cricetinae , Glicosilação , Ornitina Carbamoiltransferase/genética , Proteínas Recombinantes/isolamento & purificação , Ativador de Plasminogênio Tecidual/análise , Ativador de Plasminogênio Tecidual/isolamento & purificaçãoRESUMO
The two-kringle domain of tissue-type plasminogen activator (TK1-2) has been identified as a potent angiogenesis inhibitor by suppressing endothelial cell proliferation, in vivo angiogenesis, and in vivo tumor growth. Escherichia coli-derived, non-glycosylated TK1-2 more potently inhibits in vivo tumor growth, whereas Pichia expression system is more efficient for producing TK1-2 as a soluble form, albeit accompanying N-glycosylation. Therefore, in order to avoid immune reactivity and improve in vivo efficacy, we expressed the non-glycosylated form of TK1-2 in Pichia pastoris and evaluated its activity in vitro. When TK1-2 was mutated at either Asn(117) or Asn(184) by replacing with Gln, the mutated proteins produced the glycosylated form in Pichia, of which sugar moiety could be deleted by endoglycosidase H treatment. When both sites were replaced by Gln, the resulting mutant produced a non-glycosylated protein, NQ-TK1-2. Secreted NQ-TK1-2 was purified from the culture broth by sequential ion exchange chromatography using SP-sepharose, Q-spin, and UNO-S1 column. The purified NQ-TK1-2 migrated as a single protein band of approximately 20 kDa in SDS-PAGE and its mass spectrum showed one major peak of 19,950.71 Da, which is smaller than those of two glycosylated forms of wild type TK1-2. Functionally, the purified NQ-TK1-2 inhibited endothelial cell proliferation and migration stimulated by bFGF and VEGF, respectively. Therefore, the results suggest that non-glycosylated TK1-2 useful for the treatment of cancer can be efficiently produced in Pichia, with retaining its activity.
Assuntos
Células Endoteliais/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Kringles/genética , Pichia/genética , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/farmacologia , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/isolamento & purificação , Inibidores da Angiogênese/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Mutagênese Sítio-Dirigida , Relação Estrutura-Atividade , Timidina Quinase/genética , Timidina Quinase/isolamento & purificação , Timidina Quinase/farmacologia , Ativador de Plasminogênio Tecidual/isolamento & purificação , Fatores de Crescimento do Endotélio Vascular/farmacologiaRESUMO
The work was to explore the feasibility of protein affinity purification using ligand isolated from phage library. Reteplase was used as the model protein and a humanized semi-synthetic single chain fragment variable phage library as the source of ligand. After four rounds of biopanning, reteplase-specific phage clones were greatly enriched. The scFv gene from the best phage clone was inserted to pET-29a and expressed in E. coli Rosseta. After purification by nickel-affinity and refolding, this scFv protein was proven to recognize reteplase specifically and sensitively in ELISA and dot-blotting. Its binding constant to reteplase was 1.84x10(-8) M, measured by surface plasmon resonance. After immobilized on Sepharose 4B, the scFv was used for the affinity purification of reteplase from milk. It was found that reteplase was highly purified from the starting material. In conclusion, it has been demonstrated that humanized scFv prepared with this approach could be used as a practical affinity ligand for efficient and cost-effective purification of reteplase, as well as other therapeutic proteins.
Assuntos
Região Variável de Imunoglobulina/química , Ativador de Plasminogênio Tecidual/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia de Afinidade , Ensaio de Imunoadsorção Enzimática , Estudos de Viabilidade , Humanos , Immunoblotting , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/isolamento & purificação , Ligantes , Leite/enzimologia , Dados de Sequência Molecular , Biblioteca de Peptídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Ativador de Plasminogênio Tecidual/isolamento & purificaçãoRESUMO
A single step ion-exchange chromatography on a sulfo-propyl (SP)- Sepharose column was performed to separate both the high molecular weight (HMW)- and low molecular weight (LMW)- forms of enzymatically active urokinase type plasminogen activator from human kidney (HT1080) cell culture media. The level of urokinase secreted by the cell line reached to about 145 Plough units/ml culture broth within 48 h of cultivation. The conditioned cell culture media was applied directly to the column without any prior concentration steps. Polyacrylamide gel electrophoresis of the column eluates in the presence of sodium dodecyl sulphate showed that the cell line secretes three forms of two-chain high molecular weight (HMW) urokinase of molecular weights (M(r)) 64,000, 60,900 and 55,000. In addition, two low molecular weight (LMW) forms of M(r) 22,000 and 20,000; proteolytic cleavage products of HMW, were also found. The HMW and LMW forms had intrinsic plasminogen dependent proteolytic activity as judged by zymographic analysis. The specific activity of the pooled peak fractions increased (approximately 93-fold) to values as high as 1481 Plough units/ mg protein. Both HMW as well as LMW forms were obtained in significantly high yields.
Assuntos
Rim/citologia , Rim/metabolismo , Ativador de Plasminogênio Tecidual/isolamento & purificação , Linhagem Celular , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Humanos , Rim/enzimologia , Peso Molecular , Ativador de Plasminogênio Tecidual/química , Ativador de Plasminogênio Tipo UroquinaseRESUMO
AIM: To construct a mutant of human tissue plasminogen activator (t-PA) with activity not inhibited by plasminogen activator inhibitor-1 (PAI-1). METHODS: The finger, epidermal growth factor and kringle-1 domains were removed from native t-PA to obtain a t-PA mutant with the PAI-1 binding sites. A pUC18 plasmid containing the human t-PA full-length cDNA sequence was used as template, and the DNA sequences encoding 1-3 and 176-527 amino acids were amplified by PCR. The nucleotides AAG CAC AGG AGG (from 373 to 384) in the PAI-1 binding site changed to GCG GCC GCG GCG, so amino acid KHRR was replaced by AAAA correspondently. RESULTS: Sequencing result showed that the above t-PA mutant DNA sequence was consistent with the expected. Then it was cloned in an E.coli expression vector and was highly expressed. The expressed protein occupied about 30% of total bacterial proteins as inclusion body. After denaturation and renaturation, the active t-PA mutant was obtained,activity of which was not inhibited by PAI-1. CONCLUSION: This t-PA mutant may be developed to a new gene-engineering drug with higher therapeutic activity and lower dose requirement in the treatment of acute myocardial infarction and cerebrovascular thrombosis diseases.
Assuntos
Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/metabolismo , Sítios de Ligação/genética , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Humanos , Reação em Cadeia da Polimerase , Ligação Proteica/genética , Ativador de Plasminogênio Tecidual/isolamento & purificaçãoRESUMO
Over the past two decades tissue-type plasminogen activator (t-PA), the main physiological plasminogen activator, has been developed as a fibrin-specific thrombolytic agent for the treatment of various thromboembolic diseases. Milestones in this development include: first purification of human t-PA from uterine tissue, elucidation of the interactions regulating physiological fibrinolysis, thus providing a molecular basis for the concept of fibrin-specific plasminogen activation, first animal models of thrombosis and pilot studies in patients supporting the therapeutic potential of t-PA, cloning and expression of recombinant t-PA providing sufficient amounts for large scale clinical use, and demonstration of its therapeutic benefit in large multicenter clinical trials, mainly in patients with acute myocardial infarction (AMI), but also in patients with massive pulmonary embolism, ischemic stroke, deep vein thrombosis and peripheral arterial occlusion. Genetically modified variants of t-PA have been developed for bolus administration in patients with AMI.
Assuntos
Ativador de Plasminogênio Tecidual/história , Ativador de Plasminogênio Tecidual/uso terapêutico , Animais , Clonagem Molecular , História do Século XX , Humanos , Proteínas Recombinantes , Terapia Trombolítica , Ativador de Plasminogênio Tecidual/isolamento & purificaçãoRESUMO
Monoliths based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) can be used directly as sorbents for affinity chromatography after solid phase peptide synthesis. The quality of the synthesized products, the amount of grown peptides on a support and the reproducibility of the process must be considered. A determination of the quantity of the introducing beta-Ala (and, consequently, the total amount of synthesized peptide) was carried out. Three peptides complementary to recombinant tissue plasminogen activator (t-PA) have been synthesized using Fmoc-chemistry on GMA-EDMA disks. The peptidyl ligands were analysed by amino acid analysis, ES-MS and HPLC methods. The affinity binding parameters were obtained from frontal elution data. The results were compared with those established for GMA-EDMA affinity sorbents formed by the immobilization of the same but separately synthesized and purified ligands. The immobilization on GMA-EDMA disks was realized using a one-step reaction between the amino groups of the synthetic ligand and the original epoxy groups of monolithic material. The affinity constants found for two kinds of sorbent did not vary significantly. Finally, the directly obtained affinity sorbents were tested for t-PA separation from a cellular supernatant.
Assuntos
Compostos de Epóxi/química , Metacrilatos/química , Peptídeos/síntese química , Cromatografia de Afinidade , Estudos de Avaliação como Assunto , Espectrometria de Massas , Métodos , Ativador de Plasminogênio Tecidual/química , Ativador de Plasminogênio Tecidual/isolamento & purificação , beta-Alanina/químicaAssuntos
Eritropoetina/isolamento & purificação , Glicoproteínas/isolamento & purificação , Orosomucoide/isolamento & purificação , Eletroforese Capilar/métodos , Eritropoetina/química , Glicoproteínas/química , Humanos , Indicadores e Reagentes , Focalização Isoelétrica/métodos , Micelas , Orosomucoide/química , Peptídeos/química , Peptídeos/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrofotometria Ultravioleta , Ativador de Plasminogênio Tecidual/química , Ativador de Plasminogênio Tecidual/isolamento & purificaçãoRESUMO
Controlled shear affinity filtration (CSAF) integrates animal cell separation and product isolation in a single unit operation through the use of a specifically designed rotating disk filter with incorporated membrane chromatography column. Because of the decoupling of shear force and pressure generation and the specific hydrodynamics of the system, shear rates can be easily optimized and precisely controlled to maximize filtration performance while viability of the shear sensitive animal cells is maintained. In this study, the general methodology is demonstrated using the integration of Chinese hamster ovary cell separation and isolation of recombinant tissue plasminogen activator (t-PA) as a model example. Direct capture of t-PA from cell culture broth was realized by using custom-made affinity membranes with lysine as a robust, small molecular weight affinity ligand. Small-scale t-PA adsorption experiments, as well as microfiltration experiments, were used to design the integrated CSAF process. A Chinese hamster ovary batch culture was processed with a lab-scale prototype, yielding 86% of the t-PA in the concentrated, particle-free eluate, whereas 95% of the bulk protein was removed. Because the viability of the cells is not significantly affected and high specific flux rates can be achieved, the CSAF technology should also be well suited for continuous perfusion with integrated product isolation. A truly continuous operation could be realized with two systems in tandem configuration.