Download PDF
Semin Thromb Hemost 2001; 27(4): 325-336
DOI: 10.1055/s-2001-16886
Copyright © 2001 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel.: +1(212) 584-4662

The Production of Improved Tissue-Type Plasminogen Activator in Escherichia coli

Ralf Mattes
  • Institut für Industrielle Genetik, Universität Stuttgart, Stuttgart, Germany
Further Information

Publication History

Publication Date:
31 August 2001 (online)

    Permissions and Reprints

ABSTRACT

Tissue-type plasminogen activator (t-PA) is a valuable thrombolytic agent because of its high affinity to fibrin. When produced in mammalian cell lines, it is glycosylated, a modification that is believed to promote its rapid clearance from the circulation. Bacteria such as Escherichia coli have been tested as alternative expression systems but were not able to express the cDNA of t-PA effectively. The coding sequence for t-PA revealed a significant proportion of AGA and AGG codons, which are rarely used in the coding sequences of E. coli. The argU and argW gene products of E. coli proved to be minor tRNAarg species, respectively decoding the very rare triplets AGA/AGG and AGG for arginine. Analysis of genomic fragments from E. coli for both tRNAarg genes revealed the presence of defective, cryptic prophages integrated within the impaired tRNA genes. Cloning and supplementation of the limiting tRNA genes argU and argW on helper plasmids improved the translation of the rare AGA and AGG codons. This augmentation improved bacterial growth and enhanced t-PA production in the form of inactive inclusion bodies. This dependence on augmentation of tRNAarg4 or tRNAarg5 for improved cell growth and expression was also observed for other genes with a high content of these rare arginine codons. Construction and production of nonglycosylated t-PA in inclusion bodies in E. coli along with improvement of the subsequent renaturation and purification procedures resulted in material comparable to that derived from CHO cells. Deletion of domain-encoding segments yielded various ``muteins'' of t-PA (e.g., reteplase [rPA]) that could be produced in and activated from the purified inclusion bodies analogously. Furthermore, it was shown that rPA has an extended half-life in the circulation because of its lack of glycosylation and impaired receptor binding capability. rPA was successfully used in various clinical studies. It is a new-generation thrombolytic agent with a longer half-life and can thus be administered more conveniently as a double bolus.

KEYWORD

Tissue plasminogen activator - Escherichia coli, production - tRNA augmentation - rPA - t-PA

REFERENCES

  • 1 GISSI Investigators. Effectiveness of intravenous thrombolytic treatment in acute myocardial infarction. Gruppo Italiano per lo Studio della Streptochinasi nell'Infarto Miocardico (GISSI).  Lancet . 1986;  1 397-402
    PubMedGoogle Scholar
  • 2 ISIS-2 Investigators. Randomised trial of intravenous streptokinase, oral aspirin, both, or neither among 17,187 cases of suspected acute myocardial infarction: ISIS-2. ISIS-2 (Second International Study of Infarct Survival) Collaborative Group.  Lancet . 1988;  2 349-360
    PubMedGoogle Scholar
  • 3 Pennica D, Holmes W E, Kohr W J. Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli .  Nature . 1983;  301 214-221
    CrossrefPubMedGoogle Scholar
  • 4 Rijken D C, Hoylaerts M, Collen D. Fibrinolytic properties of one-chain and two-chain human extrinsic (tissue-type) plasminogen activator.  J Biol Chem . 1982;  257 2920-2925
    PubMedGoogle Scholar
  • 5 GUSTO Investigators. An international randomized trial comparing four thrombolytic strategies for acute myocardial infarction. The GUSTO investigators.  N Engl J Med . 1993;  329 673-682
    CrossrefPubMedGoogle Scholar
  • 6 Vehar G A, Spellman M W, Keyt B A. Characterization studies of human tissue-type plasminogen activator produced by recombinant DNA technology.  Cold Spring Harb Symp Quant Biol . 1986;  51 551-562
    PubMedGoogle Scholar
  • 7 Fisher K L, Gorman C M, Vehar G A, O'Brien D P, Lawn R M. Cloning and expression of human tissue factor cDNA.  Thromb Res . 1987;  48 89-99
    CrossrefPubMedGoogle Scholar
  • 8 Arathoon W R, Builder S E, Lubiniecki A S. Manufacture of biologically active plasminogen activator in suspension cultures of recombinant CHO cells.  U.S. Patent 5053334 1991.
    PubMedGoogle Scholar
  • 9 Wurm F M, Johnson A, Ryll T. Gene transfer and amplification in CHO cells. Efficient methods for maximizing specific productivity and assessment of genetic consequences.  Ann N Y Acad Sci . 1996;  782 70-78
    PubMedGoogle Scholar
  • 10 Werner R G, Noe W, Kopp K. Appropriate mammalian expression systems for biopharmaceuticals.  Editio Cantor Verlag Drug Res . 1998;  48(II)8 870-880
    PubMedGoogle Scholar
  • 11 Brinkmann U, Mattes R E, Buckel P. High-level expression of recombinant genes in Escherichia coli is dependent on the availability of the dnaY gene product.  Gene . 1989;  85 109-114
    CrossrefPubMedGoogle Scholar
  • 12 Robinson M, Lilley R, Little S. Codon usage can affect efficiency of translation of genes in Escherichia coli Nucleic Acids Res .  1984;  12 6663-6671
    PubMedGoogle Scholar
  • 13 Bonekamp F, Andersen H D, Christensen T, Jensen K F. Codon-defined ribosomal pausing in Escherichia coli detected by using the pyrE attenuator to probe the coupling between transcription and translation.  Nucleic Acids Res . 1985;  13 4113-4123
    PubMedGoogle Scholar
  • 14 Ikemura T. Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes.  J Mol Biol . 1981;  146 1-21
    CrossrefPubMedGoogle Scholar
  • 15 Hoekema A, Kastelein R A, Vasser M, de Boer A H. Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression.  Mol Cell Biol . 1987;  7 2914-2924
    PubMedGoogle Scholar
  • 16 Lakey D L, Voladri R K, Edwards K M. Enhanced production of recombinant Mycobacterium tuberculosis antigens in Escherichia coli by replacement of low-usage codons.  Infect Immun . 2000;  68 233-238
    PubMedGoogle Scholar
  • 17 Calogero R A, Pon C L, Gualerzi C O. Chemical synthesis and in vivo hyperexpression of a modular gene coding for Escherichia coli translational initiation factor IF1.  Mol Gen Genet . 1987;  208 63-69
    PubMedGoogle Scholar
  • 18 Hale R S, Thompson G. Codon optimization of the gene encoding a domain from human type 1 neurofibromin protein results in a threefold improvement in expression level in Escherichia coli Protein Expr Purif .  1998;  12 185-188
    PubMedGoogle Scholar
  • 19 Sharp P M, Li W H. Codon usage in regulatory genes in Escherichia coli does not reflect selection for `rare' codons.  Nucleic Acids Res . 1986;  14 7737-7749
    PubMedGoogle Scholar
  • 20 Ernst J F, Kawashima E. Variations in codon usage are not correlated with heterologous gene expression in Saccharomyces cerevisiae and Escherichia coli J Biotechnol .  1988;  7 1-10
    PubMedGoogle Scholar
  • 21 Loshon C A, Tovar-Rojo F, Goldrick S, Setlow P. The expression of a highly expressed Bacillus subtilis gene is not reduced by introduction of multiple codons normally not present in such genes.  FEMS Microbiol Lett . 1989;  53 59-63
    PubMedGoogle Scholar
  • 22 Sorensen M A, Kurland C G, Pedersen S. Codon usage determines translation rate in Escherichia coli J Mol Biol .  1989;  207 365-377
    PubMedGoogle Scholar
  • 23 Dong H, Nilsson L, Kurland C G. Co-variation of tRNA abundance and codon usage in Escherichia coli at different growth rates.  J Mol Biol . 1996;  260 649-663
    CrossrefPubMedGoogle Scholar
  • 24 Mullin D A, Garcia G M, Walker J R. An E. coli DNA fragment 118 base pairs in length provides dnaY + complementing activity.  Cell . 1984;  37 669-674
    CrossrefPubMedGoogle Scholar
  • 25 Garcia G M, Mar P K, Mullin D A, Walker J R, Prather N E. The E. coli dnaY gene encodes an arginine transfer RNA.  Cell . 1986;  45 453-459
    CrossrefPubMedGoogle Scholar
  • 26 Lindsey D F, Mullin D A, Walker J R. Characterization of the cryptic lambdoid prophage DLP12 of Escherichia coli and overlap of the DLP12 integrase gene with the tRNA gene argU J Bacteriol .  1989;  171 6197-6205
    PubMedGoogle Scholar
  • 27 Saxena P, Walker J R. Expression of argU, the Escherichia coli gene coding for a rare arginine tRNA.  J Bacteriol . 1992;  174 1956-1964
    PubMedGoogle Scholar
  • 28 Sharp P M, Cowe E, Higgins D G. Codon usage patterns in Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster and Homo sapiens; a review of the considerable within-species diversity.  Nucleic Acids Res . 1988;  16 8207-8211
    PubMedGoogle Scholar
  • 29 Nakamura Y, Gojobori T, Ikemura T. Codon usage tabulated from international DNA sequence databases: status for the year 2000.  Nucleic Acids Res . 2000;  28 292
    CrossrefPubMedGoogle Scholar
  • 30 Schenk P M, Baumann S, Mattes R, Steinbiss H H. Improved high-level expression system for eukaryotic genes in Escherichia coli using T7 RNA polymerase and rare ArgtRNAs.  Biotechniques . 1995;  19 196-200
    PubMedGoogle Scholar
  • 31 You J, Cohen R E, Pickart C M. Construct for high-level expression and low misincorporation of lysine for arginine during expression of pET-encoded eukaryotic proteins in Escherichia coli Biotechniques .  1999;  27 950-954
    PubMedGoogle Scholar
  • 32 Kleber-Janke T, Becker W M. Use of modified BL21(DE3) Escherichia coli cells for high-level expression of recombinant peanut allergens affected by poor codon usage.  Protein Expr Purif . 2000;  19 419-424
    PubMedGoogle Scholar
  • 33 Chen G T, Inouye M. Role of the AGA/AGG codons, the rarest codons in global gene expression in Escherichia coli Genes Dev .  1994;  8 2641-2652
    PubMedGoogle Scholar
  • 34 Boule J B, Johnson E, Rougeon F, Papanicolaou C. High-level expression of murine terminal deoxynucleotidyl transferase in Escherichia coli grown at low temperature and overexpressing argU tRNA.  Mol Biotechnol . 1998;  10 199-208
    PubMedGoogle Scholar
  • 35 Imamura H, Jeon B, Wakagi T, Matsuzawa H. High level expression of Thermococcus litoralis 4-alpha-glucanotransferase in a soluble form in Escherichia coli with a novel expression system involving minor arginine tRNAs and GroELS.  FEBS Lett . 1999;  457 393-396
    CrossrefPubMedGoogle Scholar
  • 36 Roche E D, Sauer R T. SsrA-mediated peptide tagging caused by rare codons and tRNA scarcity.  EMBO J . 1999;  18 4579-4589
    CrossrefPubMedGoogle Scholar
  • 37 Del Tito J B, Ward J M, Hodgson J. Effects of a minor isoleucyl tRNA on heterologous protein translation in Escherichia coli J Bacteriol .  1995;  177 7086-7091
    PubMedGoogle Scholar
  • 38 Muramatsu T, Nishikawa K, Nemoto F. Codon and amino-acid specificities of a transfer RNA are both converted by a single post-transcriptional modification.  Nature . 1988;  336 179-181
    CrossrefPubMedGoogle Scholar
  • 39 Newman J V, Burghoff R L, Pallesen L. Stimulation of Escherichia coli F-18Col- type-1 fimbriae synthesis by leuX FEMS Microbiol Lett .  1994;  122 281-287
    PubMedGoogle Scholar
  • 40 Leskiw B K, Mah R, Lawlor E J, Chater K F. Accumulation of bldA-specified tRNA is temporally regulated in Streptomyces coelicolor A3(2).  J Bacteriol . 1993;  175 1995-2005
    PubMedGoogle Scholar
  • 41 Ueda Y, Taguchi S, Nishiyama K, Kumagai I, Miura K. Effect of a rare leucine codon, TTA, on expression of a foreign gene in Streptomyces lividans Biochim Biophys Acta .  1993;  1172 262-266
    PubMedGoogle Scholar
  • 42 Ivanov I, Alexandrova R, Dragulev B, Saraffova A, AbouHaidar M G. Effect of tandemly repeated AGG triplets on the translation of CAT-mRNA in E. coli FEBS Lett .  1992;  307 173-176
    PubMedGoogle Scholar
  • 43 Seetharam R, Heeren R A, Wong E Y. Mistranslation in IGF-1 during over-expression of the protein in Escherichia coli using a synthetic gene containing low frequency codons.  Biochem Biophys Res Commun . 1988;  155 518-523
    CrossrefPubMedGoogle Scholar
  • 44 Calderone T L, Stevens R D, Oas T G. High-level misincorporation of lysine for arginine at AGA codons in a fusion protein expressed in Escherichia coli J Mol Biol .  1996;  262 407-412
    PubMedGoogle Scholar
  • 45 Forman M D, Stack R F, Masters P S, Hauer C R, Baxter S M. High level, context dependent misincorporation of lysine for arginine in Saccharomyces cerevisiae a1 homeodomain expressed in Escherichia coli Protein Sci .  1998;  7 500-503
    PubMedGoogle Scholar
  • 46 Rietsch A, Beckwith J. The genetics of disulfide bond metabolism.  Annu Rev Genet . 1998;  32 163-184
    CrossrefPubMedGoogle Scholar
  • 47 Rippmann J F, Klein M, Hoischen C. Prokaryotic expression of single-chain variable-fragment (scFv) antibodies: secretion in L-form cells of Proteus mirabilis leads to active product and overcomes the limitations of periplasmic expression in Escherichia coli Appl Environ Microbiol .  1998;  64 4862-4869
    PubMedGoogle Scholar
  • 48 Derman A I, Prinz W A, Belin D, Beckwith J. Mutations that allow disulfide bond formation in the cytoplasm of Escherichia coli Science .  1993;  262 1744-1747
    PubMedGoogle Scholar
  • 49 Bessette P H, Aslund F, Beckwith J, Georgiou G. Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm.  Proc Natl Acad Sci USA . 1999;  96 13703-13708
    CrossrefPubMedGoogle Scholar
  • 50 Rudolph R, Lilie H. In vitro folding of inclusion body proteins.  FASEB J . 1996;  10 49-56
    PubMedGoogle Scholar
  • 51 Rudolph R, Fischer S, Mattes R. Process for the activation of t-PA or IgG after genetic expression in prokaryotes.  U.S. Patent 5453363 1995.
    PubMedGoogle Scholar
  • 52 Rudolph R, Fischer S, Mattes R. Process for the activating of gene-technologically produced heterologous disulfide bridge-containing eukaryotic proteins after expression in prokaryotes.  U.S. Patent 5593865 1997.
    PubMedGoogle Scholar
  • 53 Datar R V, Cartwright T, Rosen C G. Process economics of animal cell and bacterial fermentations: a case study analysis of tissue plasminogen activator.  Biotechnology . 1993;  11 349-357
    PubMedGoogle Scholar
  • 54 Banyai L, Varadi A, Patthy L. Common evolutionary origin of the fibrin-binding structures of fibronectin and tissue-type plasminogen activator.  FEBS Lett . 1983;  163 37-41
    CrossrefPubMedGoogle Scholar
  • 55 van Zonneveld J A, Veerman H, Pannekoek H. Autonomous functions of structural domains on human tissue-type plasminogen activator.  Proc Natl Acad Sci USA . 1986;  83 4670-4674
    PubMedGoogle Scholar
  • 56 Rijken D C, Groeneveld E. Isolation and functional characterization of the heavy and light chains of human tissue-type plasminogen activator.  J Biol Chem . 1986;  261 3098-3102
    PubMedGoogle Scholar
  • 57 Holvoet P, Lijnen H R, Collen D. Characterization of functional domains in human tissue-type plasminogen activator with the use of monoclonal antibodies.  Eur J Biochem . 1986;  158 173-177
    CrossrefPubMedGoogle Scholar
  • 58 Ny T, Elgh F, Lund B. The structure of the human tissue-type plasminogen activator gene: correlation of intron and exon structures to functional and structural domains.  Proc Natl Acad Sci USA . 1984;  81 5355-5359
    PubMedGoogle Scholar
  • 59 Weidle U H, Buckel P, Mattes R. A protease-hypersensitive deletion derivative of human tissue-type plasminogen activator.  Gene . 1988;  73 439-447
    CrossrefPubMedGoogle Scholar
  • 60 Stern A, Mattes R, Buckel P, Weidle U H. Functional topology of human tissue-type plasminogen activator: characterization of two deletion derivatives and of a duplication derivative.  Gene . 1989;  79 333-344
    CrossrefPubMedGoogle Scholar
  • 61 Korninger C, Stassen J M, Collen D. Turnover of human extrinsic (tissue-type) plasminogen activator in rabbits.  Thromb Haemost . 1981;  46 658-661
    PubMedGoogle Scholar
  • 62 Kuiper J, Otter M, Rijken D C, van Berkel J T. Characterization of the interaction in vivo of tissue-type plasminogen activator with liver cells.  J Biol Chem . 1988;  263 18220-18224
    PubMedGoogle Scholar
  • 63 Rijken D C, Emeis J J. Clearance of the heavy and light polypeptide chains of human tissue-type plasminogen activator in rats.  Biochem J . 1986;  238 643-646
    PubMedGoogle Scholar
  • 64 Stern A, Kohnert U, Rudolph R, Fischer S, Martin U. Thrombolytically active non-glycosylated protein.  U.S. Patent 5223256 1993.
    PubMedGoogle Scholar
  • 65 Martin U, Kohnert U, Stern A, Popp F, Fischer S. Comparison of the recombinant Escherichia coli-produced protease domain of tissue type plasminogen activator with alteplase, reteplase and streptokinase in a canine model of coronary artery thrombolysis.  Thromb Haemost . 1996;  76 1096-1101
    PubMedGoogle Scholar
  • 66 Martin U, Fischer S, Kohnert U. Properties of a novel plasminogen activator (BM 06.022) produced in Escherichia coli Z Kardiol .  1990 (suppl 3);  79 (167-170)
    PubMedGoogle Scholar
  • 67 Kohnert U, Rudolph R, Verheijen J H. Biochemical properties of the kringle 2 and protease domains are maintained in the refolded t-PA deletion variant BM 06.022.  Protein Eng . 1992;  5 93-100
    PubMedGoogle Scholar
  • 68 Kuiper J, van de Bilt H, Martin U, van Berkel J T. Uptake, internalization and degradation of the novel plasminogen activator reteplase (BM 06.022) in the rat.  Thromb Haemost . 1995;  74 1501-1510
    PubMedGoogle Scholar
  • 69 Rijken D C, Jie A F. Interaction of reteplase with heparin. A comparison between reteplase and alteplase.  Blood Coagul Fibrinolysis . 1996;  7 561-566
    PubMedGoogle Scholar
  • 70 Bode C, Smalling R W, Berg G. Randomized comparison of coronary thrombolysis achieved with double-bolus reteplase (recombinant plasminogen activator) and front-loaded, accelerated alteplase (recombinant tissue plasminogen activator) in patients with acute myocardial infarction. The RAPID II Investigators.  Circulation . 1996;  94 891-898
    PubMedGoogle Scholar
  • 71 Bode C, Nordt T K, Peter K, Smalling R W, Runge M S, Kubler W. Patency trials with reteplase (r-PA): what do they tell us?.  Am J Cardiol . 1996;  78 16-19
    CrossrefPubMedGoogle Scholar
  • 72 GUSTO III Investigators. A comparison of reteplase with alteplase for acute myocardial infarction. The Global Use of Strategies to Open Occluded Coronary Arteries.  N Engl J Med . 1997;  337 1118-1123
    CrossrefPubMedGoogle Scholar
  • 73 Martin U, Kaufmann B, Neugebauer G. Current clinical use of reteplase for thrombolysis. A pharmacokinetic-pharmacodynamic perspective.  Clin Pharmacokinet . 1999;  36 265-276
    PubMedGoogle Scholar
  • 74 Topol E J, Ohman E M, Armstrong P W. Survival outcomes 1 year after reperfusion therapy with either alteplase or reteplase for acute myocardial infarction: results from the global utilization of streptokinase and tPA for occluded coronary arteries (GUSTO) III trial.  Circulation . 2000;  102 1761-1765
    PubMedGoogle Scholar
  • 75 Tebbe U, Graf A, Kamke W. Hemodynamic effects of double bolus reteplase versus alteplase infusion in massive pulmonary embolism.  Am Heart J . 1999;  138 39-44
    CrossrefPubMedGoogle Scholar
  • 76 Laird J R, Dangas G, Jaff M. Intra-arterial reteplase for the treatment of acute limb ischemia.  J Invasive Cardiol . 1999;  11 757-762
    PubMedGoogle Scholar
  • 77 Ouriel K, Katzen B, Mewissen M. Reteplase in the treatment of peripheral arterial and venous occlusions: a pilot study.  J Vasc Interv Radiol . 2000;  11 849-854
    PubMedGoogle Scholar
  • 78 Gurbel P A, Serebruany V L, Shustov A R. Effects of reteplase and alteplase on platelet aggregation and major receptor expression during the first 24 hours of acute myocardial infarction treatment. GUSTO-III Investigators. Global Use of Strategies to Open Occluded Coronary Arteries.  J Am Coll Cardiol . 1998;  31 1466-1473
    CrossrefPubMedGoogle Scholar
  • 79 Seyedroudbari A, Kessler E R, Mooss A N. Time to treatment and cost of thrombolysis: a multicenter comparison of tPA and rPA.  J Thromb Thrombolysis . 2000;  9 303-308
    CrossrefPubMedGoogle Scholar
  • 80 Verstraete M. Third-generation thrombolytic drugs.  Am J Med . 2000;  109 52-58
    CrossrefPubMedGoogle Scholar
      >