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1.
Yanping Geng Shengjun Wang Qingsheng Qi 《Applied and environmental microbiology》2010,76(21):7226-7230
Recombinant human tissue plasminogen activator (rPA) is a truncated version of tissue plasminogen activator (tPA), which contains nine disulfide bonds and is prone to forming inactive inclusion bodies when expressed in bacteria. To obtain functional rPA expression, we displayed the rPA on the surface of polyhydroxybutyrate (PHB) granules using phasin as the affinity tag. rPA was fused to the N terminus of the phasin protein with a thrombin cleavage site as the linker. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis showed that rPA fusion was successfully displayed on the surface of PHB granules. An activity assay indicated that the rPA fusion is active. The in vivo surface display strategy for functional rPA expression in Escherichia coli is distinct for its efficient folding and easier purification and may be expanded to the expression of other eukaryotic proteins with complex conformation.Tissue plasminogen activator (tPA) derives from a fibrinolytic system of blood vessel endothelial cells, activates plasminogen to form plasmin, and is an effective drug for thrombolytic therapy. Native tPA is composed of 527 amino acid residues with five structural domains and 17 disulfide bonds (19). Recombinant human tissue plasminogen activator (rPA) is a variant version of tPA with nine disulfide bonds, consisting of kringle 2 and serine protease domain (12). rPA was confirmed to possess enhanced capability for thrombolysis compared with that of tPA. Therefore, rPA is more beneficial for the treatment of acute myocardial infarction (17, 26, 28).Heterologous expression of tPA as well as rPA in Escherichia coli often results in the formation of the insoluble aggregates known as inclusion bodies due to the multidisulfide bonds (3). The refolding of the inclusion bodies in vitro is a long and difficult task, especially for proteins with complex conformation and multiple disulfide bonds. In order to obtain directly the functional rPA from recombinant E. coli, many approaches have been utilized: expressing the rPA gene in E. coli trxB gor ahpC* mutant strains, of which the cytoplasm is highly oxidized; fusing the rPA gene with gpIII of ΦM13 and linking to the OmpA signal sequence, through which rPA is secreted into the medium; exploiting the novel twin-arginine translocation (Tat) pathway to obtain active rPA in the periplasmic space based on its inherent properties; and cosecreting of rPA with chaperones and adding low-molecular-size medium additives to promote the formation of disulfide bonds (6, 11, 15, 25). However, the successful expression of rPA in its soluble or active form gives rise to another task: separation and purification of soluble active rPA from large amounts of other proteins in cytoplasm or medium.Normal protein purification typically involves several chromatographic steps. Each step can be costly and time-consuming (4). The development of simple and reliable methods for protein purification, which can be applied to arbitrary products, is therefore an important goal in bioseparation technology developments. One method that was recently developed is the addition of an affinity tag sequence to the target protein gene (13). It was demonstrated that heterologous proteins can be displayed actively on the surface of biopolyester granules in E. coli by fusing to the polyhydroxyalkanoate (PHA) synthase (PhaC), which serves as an affinity tag of PHA granules (21). PHA granules are carbon inclusions produced intracellularly by bacteria for coping with changing, often oligotrophic environments (1). These inclusions are composed of a hydrophobic polyester core and hydrophilic phospholipid membrane with many embedded proteins (24). Besides PhaC, phasins (namely PhaPs) are the main proteins tightly attached to the surface of polyhydroxybutyrate (PHB) granules, which can stabilize and prevent coalescence of separate PHB granules (22). Due to the inherent properties, PhaP has been used as the affinity tag in vivo to display recombinant proteins on the surface of PHB granules (5).In this study, we fused the rPA gene to the N terminus of phaP. A thrombin cleavage site was introduced between them to release rPA from PHB granules. The fusion gene was then expressed in engineered E. coli, which was conferred with the PHB production pathway by cloning the PHB biosynthesis genes. We confirmed that recombinant rPA fusion was able to be actively expressed in vivo on the surface of PHB granules. 相似文献
2.
When F9 stem cells are treated in suspension with retinoic acid, they differentiate into embryoid bodies (EBs) consisting of an inner core of undifferentiated stem cells surrounded by an outer layer of visceral endoderm (VE). When these EBs are plated onto a fibronectin (FN)-coated substrate, VE-derived parietal endoderm (PE) cells migrate onto the substrate. It has been suggested that increased levels of tPA associated with the emerging PE cells may help mediate PE outgrowth. We now show that goat anti-human tPA, an anticatalytic antibody that crossreacts with mouse tPA, and a panel of serine protease inhibitors partially inhibit PE outgrowth. Extracellular matrix (ECM) degradation analysis demonstrates that PE cell-mediated degradation of [3H]proline-labeled ECM is time- and cell concentration-dependent. A serine protease inhibitor reduced the extent of degradation, suggesting that tPA might play a role in PE outgrowth by cleaving the ECM. In support of this contention, we demonstrate that incubation of purified FN with conditioned medium plus plasminogen results in FN proteolysis. The degradation of FN is blocked by either serine protease inhibitors or goat anti-human tPA. Our data suggest that enhanced production of tPA during PE outgrowth may facilitate the migratory behavior of PE cells by mediating the degradation of ECM components such as FN. 相似文献
3.
Expression of Active Human Tissue-Type Plasminogen Activator in Escherichia coli 总被引:10,自引:0,他引:10
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The formation of native disulfide bonds in complex eukaryotic proteins expressed in Escherichia coli is extremely inefficient. Tissue plasminogen activator (tPA) is a very important thrombolytic agent with 17 disulfides, and despite numerous attempts, its expression in an active form in bacteria has not been reported. To achieve the production of active tPA in E. coli, we have investigated the effect of cooverexpressing native (DsbA and DsbC) or heterologous (rat and yeast protein disulfide isomerases) cysteine oxidoreductases in the bacterial periplasm. Coexpression of DsbC, an enzyme which catalyzes disulfide bond isomerization in the periplasm, was found to dramatically increase the formation of active tPA both in shake flasks and in fermentors. The active protein was purified with an overall yield of 25% by using three affinity steps with, in sequence, lysine-Sepharose, immobilized Erythrina caffra inhibitor, and Zn-Sepharose resins. After purification, approximately 180 μg of tPA with a specific activity nearly identical to that of the authentic protein can be obtained per liter of culture in a high-cell-density fermentation. Thus, heterologous proteins as complex as tPA may be produced in an active form in bacteria in amounts suitable for structure-function studies. In addition, these results suggest the feasibility of commercial production of extremely complex proteins in E. coli without the need for in vitro refolding. 相似文献
4.
Mia Magnusson Emma Xuchun Lu Pia Larsson Erik Ulfhammer Niklas Bergh Helena Carén Sverker Jern 《PloS one》2015,10(10)
Tissue-type plasminogen activator (t-PA), which is synthesized in the endothelial cells lining the blood vessel walls, is a key player in the fibrinolytic system protecting the circulation against occluding thrombus formation. Although classical gene regulation has been quite extensively studied in order to understand the mechanisms behind t-PA regulation, epigenetics, including DNA methylation, still is a largely unexplored field. The aim of this study was to establish the methylation pattern in the t-PA promoter and enhancer in non-cultured compared to cultured human umbilical vein endothelial cells (HUVECs), and to simultaneously examine the level of t-PA gene expression. Bisulphite sequencing was used to evaluate the methylation status, and real-time RT-PCR to determine the gene expression level. While the t-PA promoter was stably unmethylated, we surprisingly observed a rapid reduction in the amount of methylation in the enhancer during cell culturing. This demethylation was in strong negative correlation with a pronounced (by a factor of approximately 25) increase in t-PA gene expression levels. In this study, we show that the methylation level in the t-PA enhancer appears to act as a previously unrecognized switch controlling t-PA expression. Our findings, which suggest that DNA methylation is quite dynamic, have implications also for the interpretation of cell culture experiments in general, as well as in a wider biological context. 相似文献
5.
Kristen R. Kralovich Li Li Todd A. Hembrough Donna J. Webb Larry R. Karns Steven L. Gonias 《Journal of Protein Chemistry》1998,17(8):845-854
Cytokeratin 8 (CK8) is an intermediate filament protein that penetrates to the external surfaces of breast cancer cells and is released from cells in the form of soluble heteropolymers. CK8 binds plasminogen and tissue-type plasminogen activator (t-PA) and accelerates plasminogen activation on cancer cell surfaces. The plasminogen-binding site is located at the C-terminus of CK8. In this study, we prepared GST-fusion proteins which contained either 174 amino acids from the C-terminus of CK8 (CK8f) or 134 amino acids from the C-terminus of CK18 (CK18f). A third GST-CK fusion protein was identical to CK8f except that the C-terminal lysine was mutated to glutamine (CK8fK483Q). CK8f bound plasminogen; the K
D was 0.5 M. Binding was completely inhibited by ACA. CK8fK483Q also bound plasminogen, albeit with decreased affinity (K
D 1.5 M). CK18f did not bind plasminogen at all. All three fusion proteins bound t-PA equivalently, providing the first evidence that CK18 may function as a t-PA receptor. t-PA and plasminogen cross-competed for binding to CK8f. Thus, t-PA and plasminogen cannot bind to the same CK8f monomer simultaneously. Nevertheless, CK8f still promoted plasminogen activation, probably reflecting the fact that CK8f was purified in dimeric or tetrameric form. These studies demonstrate that CK8 may promote plasminogen activation by t-PA only when present in an oligomerized state. CK18 may participate in the oligomer, together with CK8, based on its ability to bind t-PA. 相似文献
6.
目的:探究依达拉奉联合重组组织型纤溶酶原激活剂(rt-PA)对脑梗死(CI)患者的疗效影响。方法:选取于2013年5月~2015年10月期间我院收治的CI患者88例,随机分为对照组和观察组各44例,对照组给予依达拉奉治疗,观察组在对照组的基础上予以rt-PA溶栓治疗,观察两组治疗后的美国国立卫生院神经功能缺损评分(NIHSS)、疗效及血清肿瘤坏死因子-α(TNF-α)和白介素-6(IL-6)、白介素-12(IL-12)水平的变化。结果:两组治疗后NIHSS评分均较治疗前显著下降,且观察组较对照组下降更加显著,差异有统计学意义(P0.05);观察组总有效率为77.27%,明显高于对照组的50.00%,差异有统计学意义(P0.05),且两组患者在治疗过程中均未出现严重的不良反应。两组治疗后血清炎性因子水平均显著降低,差异具有统计学意义(P0.05),且观察组改善程度均明显优于对照组,差异具有统计学意义(P0.05)。结论:依达拉奉联合rt-PA能显著减轻CI患者的炎症反应,改善神经功能缺损程度,值得临床推广。 相似文献
7.
已经从melanoma细胞系中成功地获得了人组织型纤溶酶原激活剂(t-PA)cDNA,在此基础上用双脱氧终止法测定了t-PAcDNA编码区全序列及3′非编码区部分序列,并发现与Pennica等发表的t-PAcDNA序列相比,有两处差异,其一是第1725位核苷酸残基为C而非A,并使此处序列与Pennica序列相比新产生一个StyI切点,但由于差异发生在密码子第三位,没有引起编码的氨基酸变化;其二是第1777位核苷酸残基(终止密码子后第4位核苷酸残基)为T而非G,使与终止密码子相隔3个核苷酸残基处产生了一个新的TGA,与Pennica序列相比此处的BstNI切点也消失了。 相似文献
8.
Wei Tang Zi-Yong Sun Ralph Pannell Victor Gurewich Jian-Ning Liu 《Protein expression and purification》1997,11(3):279-283
A system was developed to produce recombinant urokinase-type plasminogen activator inEscherichia coli.The urokinase-type plasminogen activator was produced with a 6× His-tag at the C-terminus which was shown to have the same activity, after refolding, as the wild-type protein. Purification of the recombinant protein to homogeneity (95%) was possible by one-step affinity chromatography under denaturing conditions. As a result, proteolysis by bacterial proteases during purification was avoided. A higher refolding efficiency (40%) and a higher total recovery yield (25%) of the recombinant protein were obtained by this method. 相似文献
9.
The level of an inhibitor of tissue-type plasminogen activator (t-PA) increased slowly during the early developmental stage of seeds of Erythrina caffra Thunb. Thereafter, the inhibitor increased exponentially until the seeds reached maturity. At maturity, the t-PA inhibitor levels in the cotyledons were 38 times higher than the levels at the onset of seed development. The t-PA inhibitor accumulated at a faster rate than the storage proteins, which reached a concentration 15 times higher than the protein concentration at the onset of seed development. During the imbibition and germination process, the t-PA inhibitor decreased gradually. The inhibitor kept on decreasing during the growth of the seedlings until the 10th day after imbibition, when it leveled off at 4.1% of that of the initial inhibitor concentration. The inhibitor remained at this level until the cotyledons were shed at day 22. The total protein in the cotyledons decreased at a slower rate than the inhibitor and reached a minimum concentration at day 20 of 3.6% of the initial protein concentration in the cotyledons. Callus cultures of root, shoot, leaf, and cotyledonary tissue was established and maintained on Murashige-Skoog medium supplemented with 3% sucrose, 10 micromolar benzyladenine, and 5 micromolar 2,4-dichlorophenoxyacetic acid. A shoot cell suspension culture was established on Murashige-Skoog medium supplemented with 3% sucrose, 1 micromolar benzyladenine, and 0.5 micromolar 2,4-dichlorophenoxyacetic acid (pH 5.7) and shaken at 60 revolutions per minute. The level of t-PA inhibitor in root, shoot, leaf, and cotyledonary callus was substantially lower than in the corresponding intact tissue. The t-PA inhibitor levels in the linear growth phase was higher than in the lag or stationary growth phases of the cell suspension culture. A hydrolysate of the cell walls of tomato and E. caffra Thunb, as well as polyamines and organic acids, did not increase the concentration of t-PA inhibitor in suspension cultures or intact leaf tissue of E. caffra. The t-PA inhibitor levels of suspension cultures were increased by Na2SO4 but not by I-cysteine in the nutrient medium. 相似文献
10.
Changes of ovarian tPA,uPA and PA inhibitor activities were examined in PMSG-and hCG-treatedimmature mice during periovulatory periods.The results show that 15% of the gonadotropin-treatedanimals ovulated 8 hrs after hCG administration,about 6-8 hrs earlier than in rat.It is also shownthat not only tPA activity,but also uPA activity,was regulated by gonadotropins in ovarianhomogenates and granulosa cells,and they reached maximum prior to ovulation.No measurableamount of PAI-1 activity could be detected in mouse granulosa cell conditioned medium andfollicular fluid,but considerable amount of α_2-antiplasmin,a specific inhibitor for plasmin,wasfound in follicular fluid.Cumulus-oocyte complexes contain mainly tPA.Since the ovulated eggsstill have high tPA activity,it is thought that the enzyme in the oocyte may play an important rolein implantation. 相似文献
11.
对基因工程菌株里氏木霉 (Trichodermareesei) 30 6生物合成组织型纤溶酶原激活剂 (t PA)的代谢调节机制进行了研究。在基础发酵条件下 ,L 山梨糖、D 果糖、可溶性纤维素、CMC、麦芽糖、乳糖和棉子糖等单糖及多糖对t PA生物合成都有诱导作用 ,其中L 山梨糖的诱导效果最好 ,加量以 1 .0 %为宜。葡萄糖及其中间代谢产物对t PA的生物合成产生分解代谢物阻遏作用。纤维二糖在低浓度时可以促进t PA的生物合成而在高浓度时对t PA生物合成起反馈阻遏作用 相似文献
12.
导向性纤溶酶原激活剂的研究 总被引:5,自引:0,他引:5
溶栓疗法是血栓治疗中的一种重要措施.研制具有高选择性的导向性纤溶酶原激活剂有着重大的理论意义和实用价值.采用血栓特异的单克隆抗体及其片段来介导溶栓剂已展示出较好的应用前景.双功能抗体以及同时具有抗栓,抗凝活性的小肽正逐渐拓宽人们有关导向分子研制的视野.所有这一切都将随着分子生物学技术的不断完善而付诸实现. 相似文献
13.
Plasminogen Activator Inhibitor-1 Expression by Brain Microvessel Endothelial Cells Is Inhibited by Elevated Glucose 总被引:1,自引:0,他引:1
Peter R. Kollros †Barbara A. Konkle Anne P. Ambarian Paul Henrikson 《Journal of neurochemistry》1994,63(3):903-909
Abstract: Patients with diabetes are predisposed to microvascular disease. In the retina and brain, this is characterized by neovascularization and new capillary formation. Because of the potential importance of plasmin generation in these processes, we evaluated the effect of elevated glucose concentrations on expression of plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA), and urokinase (uPA) in cultured bovine brain endothelial cells (BBEC) versus cultured bovine aortic endothelial cells (BAEC). We observed that BBEC PAI-1 mRNA levels were decreased fivefold in cells cultured in media containing 20 m M glucose compared with BBEC cultured in media with 5.5 m M glucose, whereas expression of PAI-1 mRNA in BAEC, bovine mesenteric endothelial cells, and human umbilical vein endothelial cells was not modulated under these conditions. Expression of PAI-1 protein was also inhibited by growth of BBEC in elevated glucose, but the effect was less marked than at the mRNA level. Elevated glucose did not decrease expression of PAI-1 protein by BAEC. Withdrawal of acidic fibroblast growth factor enhanced expression of PAI-1 mRNA and protein in BBEC. Expression of tPA mRNA was not affected by the glucose concentration of the medium, and uPA mRNA was not detected in our BBEC cultures. A decrease in the local tissue activity of PAI-1 by elevated glucose concentrations, with no effect on tPA or uPA expression, would lead to an increase in the plasmin activity and thereby predispose neural tissues, such as the cerebrum and retina, of diabetic patients to neovascularization. 相似文献
14.
THERE are two principal theories of the mechanism of thrombus dissolution by the fibrinolytic system. Alkjaersig et al.1 suggested that as fibrin polymerizes, plasminogen is adsorbed preferentially to the fibrin and is available in large quantities within a thrombus which is comparatively free of antiplasmin. When an activator enters the circulation it diffuses into the clot converting the plasminogen to plasmin in situ and so promotes lysis. Ambrus and Markus2, however, proposed that when plasmin forms in the circulation naturally or during infusion of an activator it is normally bound to the excess antiplasmin present in blood. They suggested that this plasmin/antiplasmin complex is reversible and dissociates in the presence of fibrin, its preferred substrate, so allowing the plasmin to bring about fibrin dissolution by “external lysis”. Neither of these theories, however, is supported by an observed phenomena. 相似文献
15.
Quantitation of plasminogen activators present in tissue may depend to a large extent on the extraction procedure used to solubilize the enzymes. Potassium thiocyanate solution is known to be an efficient solubilizer, but it can inhibit assay systems other than fibrin plates. An equally effective acetate-detergent extractant is reported here which can be used with the highly sensitive azocase inolytic assay procedure. The results indicate that a threefold increase in activator activity can be extracted from selected tissues relative to that previously reported for a phosphate-detergent extractant. The extraction medium contains 75 mM K acetate, 0.3 M NaCl, 0.1 M L-arginine, 10 mM EDTA, 0.25% Triton X-100, final pH 4.2. 相似文献
16.
Plasminogen activator enzymes have been implicated in the regulation of growth, migration, and differentiation which occur continually in normal epidermis and cyclically in the hair follicle. To elucidate further the importance of plasminogen activation in epidermal physiology, studies were conducted using mice transgenic for human plasminogen activator inhibitor 1 (PAI-1). The epidermis of the newborn (4-7 days) transgenic mice was flaky and showed delayed hair growth compared to that of their control littermates. Histologic analyses revealed a greatly thickened stratum corneum in the transgenics. By 2 weeks after birth, no differences in epidermal morphology were apparent between transgenic and control littermates. Using in situ hybridization, immunocytochemistry, and in situ reverse zymography techniques, epidermal PAI-1 expression was correlated temporally with the aberrant epidermal morphology. These data implicate plasminogen activator activity in the regulation of epidermal shedding and follicular neogenesis. 相似文献
17.
18.
Neurochemical Research - The thrombolytic activity of tissue plasminogen activator (tPA) has undisputed benefits. However, the documented neurotoxicity of tPA raises important issues. Currently,... 相似文献
19.
Fran?oise Bruyère Laurence Melen-Lamalle Silvia Blacher Beno?t Detry Anne Masset Julie Lecomte Vincent Lambert Catherine Maillard Gunilla H?yer-Hansen Leif R. Lund Jean-Michel Foidart Agnès No?l 《PloS one》2010,5(3)
The purpose of this study is to explore the function of plasminogen activator inhibitor-1 (PAI-1) during pathological lymphangiogenesis. PAI-1, the main physiological inhibitor of plasminogen activators is involved in pathological angiogenesis at least by controlling extracellular proteolysis and by regulating endothelial cell survival and migration. Protease system''s role in lymphangiogenesis is unknown yet. Thus, based on its important pro-angiogenic effect, we hypothesized that PAI-1 may regulate lymphangiogenesis associated at least with metastatic dissemination of cancer cells. To address this issue, we studied the impact of PAI-1 deficiency in various murine models of tumoral lymphangiogenesis. Wild-type PAI-1 proficient mice were used as controls. We provide for the first time evidence that PAI-1 is dispensable for tumoral lymphangiogenesis associated with breast cancers either induced by mammary carcinoma cell injection or spontaneously appearing in transgenic mice expressing the polyomavirus middle T antigen (PymT) under the control of a mouse mammary tumor virus long-terminal repeat promoter (MMTV-LTR). We also investigated inflammation-related lymphatic vessel recruitment by using two inflammatory models. PAI-1 deficiency did neither affect the development of lymphangioma nor burn-induced corneal lymphangiogenesis. These novel data suggest that vascular remodelling associated with lymphangiogenesis and angiogenesis involve different molecular determinants. PAI-1 does not appear as a potential therapeutic target to counteract pathological lymphangiogenesis. 相似文献
20.
Seeds N Mikesell S Vest R Bugge T Schaller K Minor K 《Cellular and molecular neurobiology》2011,31(6):961-967
Plasminogen activators play an important role in synaptic plasticity associated with the crossed phrenic phenomenon (CPP)
and recovery of respiratory function after spinal cord injury. A genetic approach using knockout mice lacking various genes
in the plasminogen activator/plasmin system has shown that induction of urokinase plasminogen activator (uPA) is required
during the first hour after a C2-hemisection for the acquisition of the CPP response. The uPA knockout mice do not show the
structural remodeling of phrenic motor neuron synapses characteristic of the CPP response. As shown here uPA acts in a cell
signaling manner via binding to its receptor uPAR rather than as a protease, since uPAR knockout mice or knock-in mice possessing
a modified uPA that is unable to bind to uPAR both fail to generate a CPP and recover respiratory function. Microarray data
and real-time PCR analysis of mRNAs induced in the phrenic motor nucleus after C2-hemisection in C57Bl/6 mice as compared
to uPA knockout mice indicate a potential cell signaling cascade downstream possibly involving β-integrin and Src, and other
pathways. Identification of these uPA-mediated signaling pathways may provide the opportunity to pharmacologically upregulate
the synaptic plasticity necessary for recovery of phrenic motoneuron activity following cervical spinal cord injury. 相似文献