Tissue Plasminogen Activator Variant Research Articles

COMPARISON OF THE CYTOPLASMIC AND PERIPLASMIC PRODUCTION OF RETEPLASE IN Escherichia coli

Reteplase is the recombinant type of tissue plasminogen activator variant. In this study, preplasmic and cytoplasmic (as inclusion body: IBs) production and activity of recombinant reteplase in E. coli were investigated and compared using a pET system (pET22b and pET15b). The cDNA of reteplase was cloned by polymerase chain reaction (PCR) amplification, sequenced, inserted into the vector pET 22b and pET15b, and expressed using isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant plasmid was expressed in the form of inclusion body in pET 15b and in periplasmic space in pET22b. The obtained results of inclusion body extraction from recombinant pET22b (rpET22b) and recombinant pET15b (rpET15b) plasmids using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a band of ∼39 kD. However, the obtained results of periplasmic space extraction from rpET22b plasmid showed a very weak band, while cytoplasmic expression of reteplase (pET15b) produced a strong protein band confirmed with Western blotting. Consequently, our results demonstrated that the cytoplasmic expression system is efficient for the production of reteplase protein in prokaryote systems and a high amount of reteplase was obtained from the expressed proteins in the form of IBs. The obtained activity of rpET15b plasmid showed a higher enzyme absorbance in comparison to rpET22b plasmid. This suggests rpET15b as an appropriate candidate for reteplase production.

Cloning and Expression of a Human Tissue Plasminogen Activator Variant:K2S in Escherichia coli

The DNA sequence of Kringle-2 and serine protease domains of the human tissue plasminogen activator (reteplase, K2S) was PCR amplified. This product was then cloned into the expression vector pET15b plasmid. The presence of the insert was confirmed by restriction digestion, PCR and determination of the nucleotide sequence. By using isopropyl beta-D thiogalactopyranoside (IPTG), reteplase was induced in E. coli BL21 cells and analyzed using polyacrylamide gel electrophoresis (PAGE).

Characterization of the Hepatic Disposition of Lanoteplase, a Rationally Designed Variant of Tissue Plasminogen Activator in Rodents

Lanoteplase is a recombinant mutant of tissue-type plasminogen activator (t-PA) that was developed with an aim to overcome the drawback of rapid systemic elimination of t-PA. In this study, we examined the disposition profile of lanoteplase in vivo and the kinetics of receptor-mediated endocytosis (RME) of this recombinant t-PA in vitro to kinetically characterize the mechanism(s) underlying its tissue distribution and elimination. Integration plot analysis of the initial-phase tissue distribution in rats revealed a much lower uptake clearance (CL(uptake)) of lanoteplase in the liver than that of t-PA. Rate constants for cell surface binding, internalization, and degradation of lanoteplase were also lower than those for t-PA in primary cultured rat hepatocytes. These results suggest that the improved stability of lanoteplase in vivo could be accounted for by the delay in the RME of this recombinant protein. The CL(uptake) in the liver decreased with coadministration of lactoferrin, a ligand for the low-density lipoprotein receptor-related protein (LRP) and the asialoglycoprotein (ASGP) receptors in normal mice, and in lrpap1((-/-)) mice, which have a hereditary deficiency of LRP; In contrast, CL(uptake) was not affected by mannose, whereas that of t-PA decreased with both ligands and in the lrpap1((-/-)) mice. Thus, the hepatic disposition of lanoteplase seems to be mediated by common specific receptors for t-PA, including LRP and the ASGP receptors, whereas the mannose receptor seems to be only minimally involved in the disposition of lanoteplase.

Genetic analysis of disulfide isomerization in Escherichia coli: expression of DsbC is modulated by RNase E-dependent mRNA processing.

We designed a selection strategy for the isolation of Escherichia coli mutants exhibiting enhanced protein disulfide isomerase activity. The folding of a variant of tissue plasminogen activator (v-tPA), a protein containing nine disulfide bonds, in the bacterial periplasm is completely dependent on the level of disulfide isomerase activity of the cell. Mutations that increase this activity mediate the formation of catalytically active v-tPA, which in turn cleaves a p-aminobenzoic acid (PABA)-peptide adduct to release free PABA and thus allows the growth of an auxotrophic strain. Following chemical mutagenesis, a total of eight E. coli mutants exhibiting significantly higher disulfide isomerization activity, not only with v-tPA but also with two other unrelated protein substrates, were isolated. This phenotype resulted from significantly increased expression of the bacterial disulfide isomerase DsbC. In seven of the eight mutants, the upregulation of DsbC was found to be related to defects in RNA processing by RNase E, the rne gene product. Specifically, the genetic lesions in five mutants were shown to be allelic to rne, while an additional two mutants exhibited impaired RNase E activity due to lesions in other loci. The importance of mRNA stability on the expression of DsbC is underscored by the short half-life of the dsbC transcript, which was found to be only 0.8 min at 37 degrees C in wild-type cells but was two- to threefold longer in some of the stronger mutants. These results (i) confirm the central role of DsbC in disulfide bond isomerization in the bacterial periplasm and (ii) suggest a critical role for RNase E in regulating DsbC expression.

Calcium-phosphate mediated DNA transfer into HEK-293 cells in suspension: control of physicochemical parameters allows transfection in stirred media. Transfection and protein expression in mammalian cells.

This is the first report of transient transfection of suspended cells with purified plasmid DNA in bioreactors or spinner flasks. DNA/calcium phosphate complexes were pumped or injected directly into stirred cultures of the immortalized human embryo kidney cell line 293 (HEK-293) which had been adapted to growth in suspension. We identified culture conditions suitable for this approach and modified the protocol for the generation of precipitate complexes, based on our earlier work. In order to stabilize the 'DNA-vehicle'-complex in the culture medium, we identified pH ranges and ion-concentrations which prevent dissolution or aggregation of the precipitate particles. Such conditions maintained suspended fine particles in spinners or bioreactors for up to 6 hr. During that period, cells and precipitate complexes interacted sufficiently to allow DNA transfer and subsequent expression of recombinant protein. In a simple 5 day batch process, with a starting density of 0.3 × 10(6) cells mL(-1), about 0.5 mg L(-1) of a recombinant tissue plasminogen activator variant was observed.

A variant of tissue plasminogen activator (t-PA) comprised of the Kringle 2 and the protease domain shows a significant difference in the in vitro rate of plasmin formation as compared to the recombinant human t-PA from transformed chinese hamster ovary cells

BM 06.022 is a tissue plasminogen activator (t-PA) variant comprised of the kringle 2 and the protease domain. In vivo data from animal studies provided evidence that BM 06.022 is a potent and fibrin specific fibrinolytic agent. In this study we present a detailed analysis of the in vitro rate of plasmin formation by BM 06.022. The activation of plasminogen by BM 06.022 was reduced as compared to CHO-t-PA when using CNBr-fragments of fibrinogen as a stimulator. However, a more detailed analysis revealed that the rate of plasmin formation by t-PA and BM 06.022 in the presence of CNBr-fragments of fibrinogen is dependent on the exact concentration of the stimulator because the activity-stimulator curves of both enzymes are non-parallel. In contrast, in the presence of fibrin-monomer and its plasmin derived degradation products the course of the activity-stimulator curve of BM 06.022 and t-PA was parallel. Furthermore, the concentrations of these stimulators which allowed 50% and maximal stimulation were very similar for BM 06.022 and CHO-t-PA. The rate of plasmin formation by BM 06.022 in the presence of fibrin-monomer and fibrin degradation products was lower by a factor of 2.0 and 4.3, respectively, as compared to CHO-t-PA. Furthermore, BM 06.022 and t-PA were not or only marginally stimulated by fibrinogen indicating that the systemic activation of fibrinolytic agents as it may occur during the therapy of the myocardial infarction may be due to a stimulation by fibrin degradation products rather than due to a stimulation by fibrinogen.

5156969 Tissue plasminogen activator variant with deletion of amino acids 466–470 having fibrin specific properties : John F Gill, Leonard G Presta, Mark J Zoller assigned to Genentech Inc

5156969 Tissue plasminogen activator variant with deletion of amino acids 466–470 having fibrin specific properties : John F Gill, Leonard G Presta, Mark J Zoller assigned to Genentech Inc

5156969 Tissue plasminogen activator variant with deletion of amino acids 466–470 having fibrin specific properties: John F Gill, Leonard G Presta, Mark J Zoller assigned to Genentech Inc

5156969 Tissue plasminogen activator variant with deletion of amino acids 466–470 having fibrin specific properties: John F Gill, Leonard G Presta, Mark J Zoller assigned to Genentech Inc

Secretion of active kringle-2-serine protease in Escherichia coli

Active human tissue plasminogen activator variant kringle-2-serine protease (K2 + SP domains; referred to as MB1004) was synthesized as a secreted protein in Escherichia coli, isolated, and characterized. MB1004 is a relatively large and complex protein, approximately 38 kDa in size and containing nine disulfide bonds. MB1004 without a pro region was secreted into the periplasm of E. coli by fusing the protein to the PhoA leader peptide expressed from the tac promoter. Approximately 1% (20 micrograms/L broth) of the secreted MB1004 was purified from E. coli homogenates as a soluble, active enzyme by using a combination of lysine and Erythrina inhibitor affinity chromatography. Purified MB1004 was monomeric and single-chain, and the N-terminus was identical with the predicted amino acid sequence. The specific activity of purified MB1004 from E. coli was compared against the equivalent recombinant material purified from mammalian cells that was naturally glycosylated (MB1004G) or deglycosylated after treatment with N-glycanase (MB1004N). Results from four different in vitro assays showed that MB1004 and MB1004N had similar activities. Both exhibited 4-12-fold higher specific activity than MB1004G in plasminogen activation assays. These results suggest that an inaccurate picture of specific activity can be obtained if the effects of glycosylation are not considered. By utilization of secretion in E. coli, nonglycosylated MB1004 was purified without in vitro refolding and was shown to be suitable for structure-function studies.

Carbohydrate structures of a human tissue plasminogen activator variant expressed in recombinant Chinese hamster ovary cells

The carbohydrate structures of a genetically engineered human tissue plasminogen activator variant bearing a single N-glycosylation site at Asn 448 are reported. After isolation of the tryptic glycopeptide and liberation of the N-linked carbohydrates by polypeptide: N-glycosidase F, 6 major oligosaccharide fractions were separated by HPLC on NH 2-bonded phase. Their structures were determined by compositional and methylation analyses combined with fast atom bombardment mass spectrometry. Seventy percent of the carbohydrates were of the biantennary complex type with fucose at the proximal GlcNAc and zero, one or two α2-3 linked NeuAc. The remainder were triantennary structures with one, two or three NeuAc.