Summary
To examine the secretory production of heterologous proteins by Streptomyces lividans, we fused the DNA encoding the signal peptide of the α-amylase inhibitor tendamistat, derived from S. tendae with a synthetic gene encoding the thrombin inhibitor hirudin. The analysis of secretion by immunoblots revealed an efficient translocation of hirudin through the membrane, with no detectable immunoreaction among the cellular proteins. The secreted hirudin was stable in the shaking culture for about 6 days. A comparison of the hirudin secreted by S. lividans and recombinant reference hirudin from yeast by immunoblots and thrombin inhibition assays shows that hirudin from Streptomyces has a lower specific activity, which may be due to a different aminoterminal sequence or to inexact processing of the precursor.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bender E, Koller K-P, Engels J (1990) Secretory synthesis of human interleukin-2 by Streptomyces lividans. Gene 86:227–232
Benigni R, Antonov RP, Carere LA (1975) Estimate of the genome size by renaturation studies in Streptomyces. Appl Microbiol 30:324–326
Bernan V, Filpula D, Herber W, Bibb M, Katz E (1985) The nucleotide sequence of the tyrosinase gene from Streptomyces antibioticus and characterization of the gene product. Gene 37:101–110
Bode W, Mayr I, Baumann U, Huber R, Stone SR, Hofsteenge J (1989) The refined 1.9 Å crystal structure of human α-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J 8:3467–3475
Brauer D, Engels J, Habermann P, Uhlmann E, Wengenmayer F (1985) Gentechnologisches Verfahren zur Herstellung von Hirudinen und Mittel zur Durchführung dieses Verfahrens. German patent no. DE 3429430
Chang SY, Chang S (1988) Secretion of heterologous proteins in Streptomyces lividans. In: Okami Y, Beppu T, Ogawara H (eds) Biology of actinomycetes 88 (Proceedings of the 7th International Symposium on the biology of actinomycetes). Japan Scientific Society Press, Tokyo, pp 103–107
Dodt J, Seemüller U, Maschler R, Fritz H (1985) The complete covalent structure of hirudin. Localization of the disulfide bonds. Biol Chem Hoppe-Seyler 366:379–385
Dodt J, Schmitz T, Schäfer T, Bergmann C (1986) Expression, secretion and processing of hirudin in E. coli using the alkaline phosphatase signal sequence. FEBS Lett 202:373–377
Hopwood DA, Bibb MJ, Chater KF, Kieser T, Thompson CJ, Kieser HM, Lydiate DJ, Schrempf H (1985) Genetic manipulation of Streptomyces: a laboratory manual. John Innes Foundation. Norwich, UK
Katz E, Thompson CJ, Hopwood DA (1983) Cloning and expression of the tyrosinase gene from Streptomyces antibioticus in Streptomyces lividans. J Gen Microbiol 129:2703–2714
Koller K-P, Riess G (1989) Heterologous expression of the α-amylase inhibitor gene cloned from an amplified genomic sequence of Streptomyces tendae. J Bacteriol 171:4953–4957
Koller K-P, Riess G, Sauber K, Uhlmann E, Wallmeier H (1989) Recombinant Streptomyces lividans secretes a fusion protein of tendamistat and proinsulin. Biotechnology 7:1055–1059
Laemmli UK (1970) Cleavage of structural proteins during assembly of bacteriophage T4. Nature 227:680–685
Laminet AA, Plückthun A (1989) The precursor of β-lactamase: purification, properties and folding kinetics. EMBO J 5:1469–1477
Lichenstein H, Brawner ME, Miles M, Meyers CA, Young PR, Simon PL, Eckhardt T (1988) Secretion of interleukin-1β and Escherichia coli galactokinase by Streptomyces lividans. J Bacteriol 170:3924–3929
Loison G, Findeli A, Nguyen-Juilleret SB, Marquet M, Riehl-Bellon N, Carvallo D, Guerra-Santos L, Brown SW, Courtney M, Roitsch C, Lemoine Y (1988) Expression and secretion in S. cerevisiae of biologically active leech hirudin. Biotechnology 6:72–77
MacNeil DJ (1986) A flexible boiling procedure for isolating plasmid DNA from Gram positive microorganisms. J Microbiol Methods 5:115–123
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N Y.
Meyer DI (1988) Preprotein conformation: the year's major theme in translocation studies. TIBS 13:471–474
Noack D, Geuther R, Tonew M, Breitling R, Behnke D (1988) Expression and secretion of interferon-α1 by Streptomyces lividans: use of staphylokinase signals and amplification of the neo gene. Gene 68:53–62
Riehl-Bellon N, Carvallo D, Acker M, Dorsselaer A van, Marquet M, Loison G, Lemoine Y, Brown SW, Courtney M, Roitsch C (1989) Purification and biochemical characterization of recombinant hirudin produced by Saccharomyces cerevisiae. Biochemistry 28:2941–2949
Sanger F, Coulson AR, Barnell BG, Smith AJH, Roe BA (1980) Single stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol 143:161–178
Scharf M, Engels J, Tripier D (1989) Primary structures of new ‘isohirudins’. FEBS Lett 255:105–110
Towbin H, Staehlin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354
Zsebo KM, Lu H-S, Fieschko JC, Goldstein L, Davis J, Duker K, Suggs SV, Lai P-H, Bitter GA (1986) Protein secretion from Saccharomyces cerevisia directed by the prepro-α-factor leader region. J Biol Chem 255:7973–7977
Author information
Authors and Affiliations
Additional information
Offprint requests to: J. Engels
Rights and permissions
About this article
Cite this article
Bender, E., Vogel, R., Koller, KP. et al. Synthesis and secretion of hirudin by Streptomyces lividans . Appl Microbiol Biotechnol 34, 203–207 (1990). https://doi.org/10.1007/BF00166781
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00166781