Secretion of active recombinant human tissue plasminogen activator derivatives in Escherichia coli

The DNA fragment coding for kringle 2 plus serine protease domains (K2S) of tissue plasminogen activator (tPA) was inserted into a phagemid vector, pComb3HSS. In the recombinant vector, pComb3H-K2S, the K2S gene was fused to gpIII of PhiM13 and linked to the OmpA signal sequence. The resulting gene, rK2S-gpIII, was inducibly expressed in Escherichia coli XL-1 Blue. The protein was presented on the phage particle. To stop the expression of gpIII, a stop codon between K2S and the gpIII gene was inserted by site-directed mutagenesis. This mutated vector, MpComb3H-K2S, was transformed in XL-1 Blue. After induction with IPTG (isopropyl-beta-D-thiogalactopyranoside), rK2S was found both in the periplasm as an inactive form of approximately 32% and in the culture supernatant as an active form of approximately 68%. The secreted form of rK2S was partially purified by ammonium sulfate (55%) precipitation. The periplasmic form was isolated from whole cells by chloroform extraction. The fibrin binding site of kringle 2 was demonstrated in all expressed versions (phage-bound, periplasmic, and secreted forms) using the monoclonal anti-kringle 2 antibody (16/B). Only the secreted form of rK2S revealed a fibrinogen-dependent amidolytic activity with the specific activity of 236 IU/microg. No amidolytic activity of rK2S was observed in either the periplasmic or the phage-bound form. The secretion of rK2S as an active enzyme offers a novel approach for the production of the active-domain deletion mutant tPA, rK2S, without any requirements for bacterial compartment preparation and in vitro refolding processes. This finding is an important technological advance in the development of large-scale, bacterium-based tPA production systems.     

High-level synthesis of biologically active human plasminogen activator inhibitor type 1 (PAI-1) in Escherichia coli

Segments of a cDNA encoding human plasminogen activator inhibitor type 1 (PAI-1) were subcloned into a highly regulated and inducible Escherichia coli expression system. A plasmid encoding the mature form of human endothelial PAI-1 produced a functional recombinant molecule, as indicated by its ability to inhibit tissue plasminogen activator's enzymatic activity. In contrast to PAI-1 isolated from human fibrosarcoma cells, the biological activity of the recombinant PAI-1 was not dependent on pretreatment with denaturing agents. A construct encoding a polypeptide lacking the first 80 amino acids of PAI-1 also produced elevated levels of the truncated recombinant protein. However, this truncated product was functionally inactive, indicating that an intact N terminus is required for activity.     

Purification and characterization of tissue plasminogen activator kringle-2 domain expressed in Escherichia coli

We have expressed the 174-263 fragment (kringle-2 domain) of human tissue-type plasminogen activator (t-PA) in Escherichia coli by secretion into the periplasmic space using the alkaline phosphatase promoter and stII enterotoxin signal sequence. A large portion of the secreted protein is associated with an insoluble cellular fraction. This material can be solubilized by extraction with denaturant and reducing agent and then recovered in active form by refolding in the presence of reduced and oxidized glutathione. Kringle-2 is then easily purified by affinity chromatography on lysine-Sepharose followed by cation-exchange chromatography. The isolated protein has an amino acid composition and N-terminal sequence as expected for the 174-263 fragment of t-PA, indicating that the signal peptide has been properly removed. Circular dichroic spectra suggest that the protein is folded similar to the kringle-4 domain of plasminogen [Castellino et al. (1986) Arch. Biochem. Biophys. 247, 312-320]. Equilibrium dialysis experiments indicate a single binding site on kringle-2 for L-lysine having a KD of 100 microM. Using a method based on elution of kringle from lysine-Separose with omega-aminocarboxylic acids [Winn et al. (1980) Eur. J. Biochem. 104, 579-586], we have shown the lysine binding site of t-PA kringle-2 to have a preference for a ligand with 8.8-A separation between amine and carboxylate functions. Charge interactions with the epsilon-amino group of L-lysine are important in binding since the affinities for N epsilon-acetyl-L-lysine, L-arginine, and gamma-guanidinobutyric acid are decreased greater than 2000-fold, 200-fold, and 12-fold, respectively, relative to the affinity for L-lysine.(ABSTRACT TRUNCATED AT 250 WORDS)     

人组织型纤溶酶原激活剂突变体微小基因的构建

tPA基因全长约36kb,至少由13个内含子分隔为14个外显子。根据tPA的第一、二外显子的编码情况,考虑建立从第二至第六外显子序列在内的tPA微小基因。即将tPA的部分基因组序列与LAtPA cDNA的序列在第六外显子的NarI位点处相连。     

Expression of biologically active kringle 5 domain of human plasminogen in Escherichia coli

The kringle 5 domain of plasminogen exhibits potent inhibitory effect on endothelial cell proliferation. It can also cause cell cycle arrest and apoptosis of endothelial cell specifically, and shows promise in anti-angiogenic therapy. It has been prepared via both proteolysis of native plasminogen and recombinant DNA methodologies. When previously expressed in Escherichia coli, recombinant kringle 5 mainly deposited as inactive, insoluble inclusion bodies and the refolding yield was low. In the present study, human kringle 5 was fusion-expressed with GST (gluthathione-S-transferase) under the control of T7 promoter in E. coli. The IPTG-induced GST-kringle 5 was about 20% of the total cellular proteins and, among the expressed GST-kringle 5 proteins, 80% was present in the supernatant. The GST-kringle 5 fusion protein exhibited some anti-proliferation activity towards bovine capillary endothelial cells. After GST-kringle 5 purification, subsequent enterokinase release of intact kringle 5 from the fusion protein and further purification by gluthathione-Sepharose 4B affinity chromatography, the recombinant kringle 5, with a yield of 10.5 mg/L culture, displayed apparent inhibition of endothelial cell proliferation in a dose-dependent manner with ED50 about 20 nM.     

Functional properties of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli

The kringle-2 domain (residues 176-262) of tissue-type plasminogen activator (t-PA) was cloned and expressed in Escherichia coli. The recombinant peptide, which concentrated in cytoplasmic inclusion bodies, was isolated, solubilized, chemically refolded, and purified by affinity chromatography on lysine-Sepharose to apparent homogeneity. [35S]Cysteine-methionine-labeled polypeptide was used to study the interactions of kringle-2 with lysine, fibrin, and plasminogen activator inhibitor-1. The kringle-2 domain bound to lysine-Sepharose and to preformed fibrin with a Kd = 104 +/- 6.2 microM (0.86 +/- 0.012 binding site) and a Kd = 4.2 +/- 1.05 microM (0.80 +/- 0.081 binding site), respectively. Competition experiments and direct binding studies showed that the kringle-2 domain is required for the formation of the ternary t-PA-plasminogen-intact fibrin complex and that the association between the t-PA kringle-2 domain and fibrin does not require plasmin degradation of fibrin and exposure of new COOH-terminal lysine residues. We also observed that kringle-2 forms a complex with highly purified guanidine-activated plasminogen activator inhibitor-1, dissociable by 0.2 M epsilon-aminocaproic acid. The kringle-2 polypeptide significantly inhibited tissue plasminogen activator/plasminogen activator inhibitor-1 interaction. The kringle-2 domain bound to plasminogen activator inhibitor-1 in a specific and saturable manner with a Kd = 0.51 +/- 0.055 microM (0.35 +/- 0.026 binding site). Therefore, the t-PA kringle-2 domain is important for the interaction of t-PA not only with fibrin, but also with plasminogen activator inhibitor-1 and thus represents a key structure in the regulation of fibrinolysis.     

cDNA cloning and expression in Escherichia coli of a plasminogen activator inhibitor from human placenta

Two nearly full-length cDNAs for placental plasminogen activator inhibitor (PAI) have been isolated from a human placenta lambda gt11 cDNA library. One positive (lambda PAI-75.1) expressed a protein that could adsorb and purify anti-PAI antibodies. The expressed protein inhibited the activity of human urokinase in a fibrin autography assay, and formed a 79-kDa (reduced) covalent complex with 125I-urokinase that could be immunoprecipitated with anti-PAI. The cDNA insert of the longer isolate (lambda PAI-75.15) consisted of 1909 base pairs, including a 5'-noncoding region of 55 base pairs, an open reading frame of 1245 base pairs, a stop codon, a 3'-noncoding region of 581 base pairs, and a poly(A) tail. The size of the mRNA was estimated to be 2.0 kilobases by Northern blot analysis. The translated amino acid sequence consisted of 415 amino acids, corresponding to a 46.6-kDa protein. The sequence was related to members of the serpin gene family, particularly ovalbumin and the chicken gene Y protein. Like these avian proteins, placental PAI appears to lack a cleavable NH2-terminal signal peptide. Residues 347-376 were identical to the partial sequence reported recently for a PAI isolated from the human monocytic U-937 cell line. Placental PAI mRNA was apparently expressed at low levels in human umbilical vein endothelial cells, but was not detectable in HepG2 hepatoma cells. It was present in U-937 cells and was inducible at least 10-fold by phorbol 12-myristate 13-acetate. Thus placental PAI is a unique member of the serpin gene family, distinct from endothelial-type PAI. It is probably identical to monocyte-macrophage PAI.     

Disulfide pairing of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli.

The kringle-2 domain of tissue plasminogen activator, cloned and expressed in Escherichia coli (Wilhelm, O.G., Jaskunas, S.R., Vlahos, C.J., and Bang, N.U. (1990) J. Biol. Chem. 265, 14606-14611), was internally radiolabeled using [35S]methionine-cysteine. Following refolding and isolation, the labeled polypeptide was further purified by reverse-phase high performance liquid chromatography. The purified kringle-2 domain was digested with thermolysin, and the resulting peptides were purified by high performance liquid chromatography. Five major peptides containing 35S were obtained. Amino acid sequence analysis showed that these peptides represented various cleavage products containing one or more of the following disulfides: Cys180-Cys261, Cys201-Cys243, Cys232-Cys256 (sequence numbering based on Pennica et al. (Pennica, D., Holmes, W.E., Kohr, W.J., Hakins, R.N., Vehar, G. A., Ward, C.A., Bennett, W.F., Yelverton E., Seeburg, P.H., Heynecker, H.L., Goeddel, E.V., and Collen, D. (1983) Nature 301, 214-221)). These results confirm that the refolding methodology used produced kringle-2 with the predicted disulfide linkage and, thus, yielded material suitable for structural and functional studies.     

Purification of active human plasminogen activator inhibitor 1 from Escherichia coli. Comparison with natural and recombinant forms purified from eucaryotic cells

Plasminogen activator inhibitor 1 (PAI-1) inhibits both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) and, therefore, is an important regulator of plasminogen activation. We have developed eucaryotic and procaryotic expression systems for PAI-1 and characterized the recombinant glycosylated and non-glycosylated products, together with a non-recombinant natural control, produced in the histosarcoma cell line HT 1080. For eucaryotic expression, the PAI-1 cDNA was stably transfected into chinese hamster ovary cells (CHO cells), while procaryotic expression in Escherichia coli was examined after inserting the DNA sequence encoding the mature PAI-1 protein into an inducible expression vector. Recombinant PAI-1 from CHO cells was purified approximately 50-fold in two steps and was indistinguishable from natural PAI-1. Between 3% and 4% of total cellular protein in the procaryotic expression system consisted of PAI-1, from which it was purified approximately 30-fold, with yields of between 15% and 20%. This PAI-1 formed 1:1 complexes with uPA and also with the single- and two-chain forms of tPA. Kinetic analysis demonstrated that the procaryote-produced PAI-1 had an inhibitory activity towards all three forms of PA that resembled that of natural PAI-1 with association rate constants of approximately 10(7) M-1 s-1. In contrast to PAI-1 from eucaryotic cells, the PAI-1 from E. coli had an inherent activity equal to that of guanidine/HCl-activated natural PAI-1. The activity could not be increased by treatment with denaturants suggesting that the latent form of PAI-1 was absent. However, at 37 degrees C the procaryote-produced PAI-1 lost activity at the same rate as natural PAI-1, with approximately 50% of the activity remaining after 3 h. This activity could be partially restored by treatment with 4 M guanidine/HCl. E. coli-derived PAI-1, added to human plasma and fractionated by Sephacryl S-200 chromatography, eluted in two peaks that were similar to those obtained with guanidine-activated PAI-1 from eucaryotic cells, suggesting that it bound to the PAI-1-binding protein (vitronectin).     

Purification and characterization of active and latent forms of recombinant plasminogen activator inhibitor 1 produced in Escherichia coli.

Plasminogen activator inhibitor 1 (PAI-1), the principal physiological inhibitor of tissue plasminogen activator (tPA), is a protein of 379 amino acids and belongs to the SERPIN family of serine protease inhibitors. We have previously described methods to express [Sisk et al. (1990) Gene 96, 305-309] and purify [Reilly et al. (1990) J. Biol. Chem. 265, 9570-9574] a highly active form of the protein in substantial amounts, from Escherichia coli. Further analyses of this material showed the presence of small but significant amounts of latent rPAI-1. The present paper describes for the first time purification of latent and active forms of rPAI-1 from a single preparation, as well as the functional and structural characteristics of the two forms. Latent rPAI-1, which has properties similar to the latent forms described by other groups, was separated from active rPAI-1 by high-resolution ion-exchange chromatography or by affinity chromatography using immobilized anhydrotrypsin. It had low intrinsic activity (< 5% of active rPAI-1) and was partially reactivated by guanidine hydrochloride treatment or by incubation with vitronectin. Conversion of the active rPAI-1 to the latent form was influenced by temperature and additives including sucrose, EDTA, and arginine. Active and latent rPAI-1 did not show any obvious differences in their primary structures and displayed remarkably similar secondary structures as determined by circular dichroism spectral analyses. However, they did exhibit differences in tryptophan fluorescence, suggesting tertiary structural differences between the two forms.     

Novel biomimetic affinity ligands for human tissue plasminogen activator

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.     

Purification and characterization of recombinant plasminogen activator inhibitor-1 from Escherichia coli

A recombinant form of plasminogen activator inhibitor-1 (rPAI-1) has been purified from lysates of pCE1200, a bacterial expression vector containing the full length PAI-1 gene, by utilizing sequential anion exchange and cation exchange chromatography on Q-Sepharose and S-Sepharose columns. Approximately 140 mg of rPAI-1, estimated at 98% purity on the basis of analytical high performance liquid chromatography, could be obtained from 200 g wet weight of cells. The purified protein exhibited a single Coomassie Blue-stainable band at the region of Mr = 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an NH2-terminal amino acid sequence consistent with the expected translation product of the pCE1200 PAI-1 insert. The rPAI-1 rapidly inhibited single- and two-chain tissue plasminogen activators, as well as urokinase, with apparent second order rate constants in the range of 2-5 x 10(7) M-1 s-1. A specific activity measurement of 250,000 units/mg was calculated for the rPAI-1 based on its ability to inhibit the enzymatic activity of a single-chain tissue plasminogen activator. Stability studies showed that the activity of the rPAI-1 was very stable when stored at temperatures of 25 degrees C or lower, but decayed within hours when stored at 37 degrees C. Sodium dodecyl sulfate treatment, which partially activates the latent form of natural PAI-1, inactivated rPAI-1. These results show that the purified rPAI-1 produced from pCE1200 displays many of the properties associated with the biologically active form of natural PAI-1.     

Role of tissue plasminogen activator and plasminogen activator inhibitor polymorphism in myocardial infarction

A case–control association study on 229 Myocardial Infarction (MI) patients and 217 healthy controls was carried out to determine the role of tissue-plasminogen activator (t-PA) (Alu-repeat insertion (I)/deletion (D)) and plasminogen activator inhibitor (PAI-1) (4G/5G insertion/deletion) polymorphisms with MI in the Pakistani population. In MI patients the genotype distribution of the PAI-1 gene was not found to be different when compared with the unaffected controls (P > 0.05, χ² = 1.03). The risk allele 4G was also not associated with MI (P > 0.05, χ² = 0.46, odds ratio (OR) = 1.1 (95% confidence interval (CI) = 0.84–1.43), P > 0.05). Similarly, the genotype frequencies of t-PA I/I, I/D and D/D were not different from the unaffected controls (P > 0.05, χ² = 1.60), and the risk allele “I” was not found to be associated with MI (P > 0.05, χ² = 1.35, OR = 0.86 (95% CI = 0.66–1.11), P > 0.05). However, when the data were distributed along the lines of gender a significant association of the 4G/4G PAI-1 genotype was observed with only the female MI patients (P < 0.05, z-test = 2.21). When the combined genotypes of both the polymorphisms were analyzed, a significant association of MI was observed with the homozygous DD/4G4G genotype (P < 0.01, z-test = 2.61), which was specifically because of the female samples (P = 0.01, z-test = 2.53). In addition smoking (P < 0.001, χ² = 13.52, OR = 3.45 (95% CI = 1.77–6.94)), diabetes (P < 0.001, χ² = 22.45, OR = 8.89 (95% CI = 2.96–29.95)), hypertension (OR = 7.76 (95% CI = 2.88–22.68), P < 0.001) family history (P < 0.001, χ² = 13.72, OR = 3.7 (95% CI = 1.71–8.18)) and lower HDL levels (P < 0.05) were found to be significantly associated with the disease. In conclusion the PAI-1 gene polymorphism was found to have a gender specific role in the female MI patients.     

The regulation of tissue plasminogen activator activity by human fibroblasts

We have found that live and ethanol-fixed fibroblasts, when covered with conditioned medium containing tissue plasminogen activator, associate with the enzyme and remove it from the medium. Binding of tissue plasminogen activator to fixed cells showed equilibrium kinetics with maximal uptake corresponding to 2.4 units of enzyme per 10(6) fixed cells. Enzyme bound to fixed cells could activate plasminogen and produce plaques of caseinolysis in casein-plasminogen-agar overlays. Electrophoretic analysis showed it covalently attached to a fibroblast component with a molecular weight of 40,000-50,000. Sequestration of tissue plasminogen activator by live fibroblasts showed nonsaturable first order kinetics with a rate constant of 0.465/hr. We conclude that active enzyme is bound to a surface receptor, then internalized and degraded. Fibroblasts did not release the binding molecule into the medium; binding of tissue plasminogen activator from the medium was unaffected by heparin or thrombin. This phenomenon differs from that described by Baker et al. and ascribed to "proteasenexin."     

Increased expression of recombinant human tissue plasminogen activator in Leishmania tarentolae

Recombinant tissue plasminogen activator (rt-PA) is one of the most important thrombolytic agents for treating cardiovascular obstructions such as stroke. Glycoprotein rt-PA is a serine protease, consisting of 527 amino acids of which 35 are cysteine residues. A variety of recombinant protein expression systems have been developed for heterologous gene expression in prokaryotic and eukaryotic hosts. In recent years, Leishmania tarentolae has been considered because of its safety aspects and special attributes in expression of complex proteins. In this study, two expression cassettes, each one including two copies of t-PA cDNA, were used for integration into the L. tarentolae genome by electroporation. Transformed clones were selected in the presence of appropriate antibiotics. Expression of active rt-PA was confirmed by Western blot and Zymography tests. Real-time PCR analysis was applied to investigate the presence of multiple t-PA gene copies in the parasite genome. Correlation of t-PA gene dosage and production rate was confirmed with real-time PCR. It was shown that the expression level of rt-PA in L. tarentolae is at least 480 IU/mL of culture media. This concentration of rt-PA is seven times higher than what was reported in previous studies in L. tarentolae and some other eukaryotic systems.     

Covalent-display of an active chimeric-recombinant tissue plasminogen activator on polyhydroxybutyrate granules surface

Objective

To develop a deliberately engineered expression and purification system for an active chimeric-recombinant tissue plasminogen activator (crtPA) using co-expression with polyhydroxybutyrate (PHB) operon genes.

Results

Fusion of crtPA with PhaC-synthase simplified the purification steps through crtPA sedimentation with PHB particles. Moreover, the covalently immobilized crtPA was biologically active as shown in a chromogenic assay. Upon WELQut-protease activity, the released single-chain crtPA converted to the two-chain form which produced a pattern of bands with approx. MW of 32 and 11 kDa in addition to the full length crtPA.

Conclusion

Fusion of crtPA with PhaC-synthase not only simplifies purification from the bacterial host lysate, but also co-expression of PHB operon genes creates an oxidative environment, thereby reducing the inclusion body formation possibility. The isolated crtPA-PHB granules exhibited crtPA serine protease activity. Thus, fusion with the PhaC protein could be used as a scaffold for covalent displaying of functional disulfide-rich proteins.

     

Purification of human tissue plasminogen activator with Erythrina trypsin inhibitor

Seeds of the legume Erythrina latissima contain a 20,000-dalton, single-chain protein that has been shown to inhibit the amidolytic activity of trypsin and tissue plasminogen activator. It had no comparable effect on urokinase. IC50 values of 1.1 X 10(-7) M for tissue plasminogen activator and 6.9 X 10(-10) M for trypsin were determined by titration. When coupled to agarose, the Erythrina inhibitor provided an effective reagent for affinity purification of tissue plasminogen activator from melanoma cell-conditioned tissue culture medium. Using this as a single-step procedure, 270-fold purified enzyme was reproducibly obtained with yields of 90% or greater. Both one- and two-chain forms of tissue plasminogen activator were purified. The enzyme migrated, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as a predominant 72,000-dalton doublet with lesser amounts of immunochemically similar, 115,000- and 68,000-dalton components.