Isolation of a human tissue-type plasminogen-activator genomic DNA clone and its expression in mouse L cells

Browne, Michael; TYRRELL, A. W. R.; Chapman, C.G.; Carey, J.E.; Glover, David M.; Grosveld, Frank; Dodd, Ian B.; Robinson, Jan

doi:10.1016/0378-1119(85)90235-5

Cited by 33 publications

(8 citation statements)

References 23 publications

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“…This process due to formation of antigen-antibody complex, confirmed the exclusive presence of t-PA in the sample [40]. The low-molecular-mass bands may be due to limited proteolysis of the protein [41].…”

Section: Discussionsupporting

confidence: 59%

Cloning and Expression of Functional Full-Length Human Tissue Plasminogen Activator in Pichia pastoris

Majidzadeh‐A

Khalaj

Davami

et al. 2010

Appl Biochem Biotechnol

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Human tissue plasminogen activator (t-PA) plays a pivotal role in the treatment of acute myocardial infarction, ischemic stroke, and deep vein thrombosis. It has the benefit of generating no adverse effects such as fibrinogen depletion, systemic hemorrhage, and immunologic reactions. Human t-PA is a serine-protease enzyme containing 527 amino acid residues in five structural domains. The correct folding of t-PA requires the correct pairing of 17 disulfide bridges in the molecule. A gene encoding full-length human t-PA was cloned into pPICZαA expression vector downstream of alcohol oxidase promoter and α-mating signal sequence from Saccharomyces cerevisiae and flush with the kex2 cleavage site to express the protein with a native N terminus. The methylotrophic yeast, Pichia pastoris GS115 strain, was transformed with this cassette, and methanol utilizing (mut+) transformants were selected for production and secretion of human t-PA into culture media. SDS-PAGE and Western blot analysis showed the expressed bands of t-PA protein. Zymography test indicated suitable folding and proper function of the expressed recombinant human t-PA in conversion of plasminogen to plasmin and gelatin lysis. Amidolytic activity test showed the amidolytic activity of 1,650 IU/ml. The results of this study concluded that P. pastoris methylotrophic yeast can be a suitable alternative for mammalian and prokaryotic expression systems to produce t-PA.

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Section: Discussionsupporting

confidence: 59%

Cloning and Expression of Functional Full-Length Human Tissue Plasminogen Activator in Pichia pastoris

Majidzadeh‐A

Khalaj

Davami

et al. 2010

Appl Biochem Biotechnol

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“…Zymography of culture supernatants of transformed Leishmania with pF4X1.4sat‐tPA and pF4X1.4hyg‐tPA, and both showed three distinct bands, corresponding to 25, 30 and 65 kDa. The lower‐molecular‐mass bands may have resulted from limited proteolysis of the tPA [36,37].…”

Section: Discussionmentioning

confidence: 99%

Expression of human tissue plasminogen activator in the trypanosomatid protozoan Leishmania tarentolae

Soleimani¹,

Mahboudi²,

Davoudi³

et al. 2007

Biotech and App Biochem

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A variety of recombinant protein expression systems have been developed for heterologous genes in both prokaryotic and eukaryotic systems such as bacteria, yeast, mammals, insects, transgenic animals and transgenic plants. Also, it has been reported that Leishmania tarentolae, a trypanosomatid protozoan parasite of the white-spotted wall gecko (Tarentola annularis), has the capability of expressing heterologous genes. Trypanosomatidae are rich in glycoproteins, which can account for more than 10% of total protein. The oligosaccharide structures of their glycoproteins are similar to those of mammals with N-linked galactose, and sialic acid residues. For a variety of reasons, including the glycosylation patterns and the secondary structures of some of these proteins, synthesis in eukaryotic system is highly preferable. In addition, formation of native disulfide bonds in complex eukaryotic proteins is tremendously important. In the present study, we tried to express the tPA (tissue plasminogen activator) gene in L. tarentolae. This protein is a thrombolytic agent with 527 amino acid residues. tPA possesses serine-protease activity, with 35 cysteine residues that participate in the formation of 17 disulfide bonds. We have used an expression cassette, including the alpha intergenic regions of Leishmania major and two sites at the 3'- and 5'-ends, for homologous recombination in L. tarentolae, in addition to antibiotic-resistant genes. Southern-blot analysis showed that the human tPA gene had been inserted into the genome of the parasite. The expression of the tPA at the mRNA and protein levels was confirmed. It was shown that the expressed tPA in this system was 70 i.u. (international units)/ml of culture media, which is much higher than levels reported previously in other systems.

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“…Tissue-type plasminogen activator was purified essentially as described (10). Preparations of Z-chain t-PA were reconstituted at 2 ^g t-PA protein/ml in 0.05 M TLis, 0.1 M NaCl, 25% (v/v) glycerol pH 9.0.…”

Section: Isolation Of T-pa B-chainmentioning

confidence: 99%

Isolation and Preliminary Characterisation of Active B-Chain of Recombinant Tissue-Type Plasminogen Activator

1986

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SummaryPurified 2-chain recombinant tissue-type plasminogen activator (t-PA) was reduced under mild conditions - 10 mM dithiothreitol/ 5° C/1.5 h - and the two chains were separated by chromatography on lysine Sepharose. The t-PA B chain was fully active as determined by its activity towards the chromogenic substrate S-2288 (H-D-ile-pro-arg p-nitroanilide). Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing or non-reducing conditions revealed a single polypeptide at Mr = 35,000 or 29,000 respectively. In addition, under non-reducing conditions a fibrinolytic band at apparent Mr = 29,000 was present after fibrin zymography. The N-terminal sequence was confirmed as ile-lys-gly. The t-PA B chain had a specific amidolytic activity, using S-2288, of 170,000 to 210,000 SU/mg protein. (This compares to a specific activity of the native 2-chain t-PA of 170,000 SU/mg). It resembles urokinase-type plasminogen activator in its inability to be stimulated by fibrin and its dose response on human fibrin plates. However, t-PA B-chain was stimulated to almost the same extent as t-PA by poly-D-lysine. The isoelectric points, at pH 5.6 and 5.7, fall outside the range generally quoted for t-PA preparations (pH 7.8-8.8).

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Isolation of a human tissue-type plasminogen-activator genomic DNA clone and its expression in mouse L cells

Cited by 33 publications

References 23 publications

Cloning and Expression of Functional Full-Length Human Tissue Plasminogen Activator in Pichia pastoris

Cloning and Expression of Functional Full-Length Human Tissue Plasminogen Activator in Pichia pastoris

Expression of human tissue plasminogen activator in the trypanosomatid protozoan Leishmania tarentolae

Isolation and Preliminary Characterisation of Active B-Chain of Recombinant Tissue-Type Plasminogen Activator

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