1887

Abstract

To study the effect of inserted peptides on the secretion and processing of exported proteins in and , pBR322-derived DNA fragments coding for small peptides were inserted between the DNA coding for the 31 amino acid α-amylase signal peptide and that coding for the mature part of the extracellular thermostable α-amylase of . Most of the inserted peptides (21 to 65 amino acids) decreased the production of the enzyme in and , the effect of each peptide being similar in the two strains. In contrast, with one peptide (a 21 amino acid sequence encoded by the extra DNA in pTUBE638), the production of α-amylase was enhanced more than 1·7-fold in in comparison with that of the parent strain. The molecular masses of the thermostable α-amylases in the periplasm of the transformants varied for each peptide insert, whereas those in the culture supernatants of the transformants had molecular masses similar to that of the mature enzyme. Based on the NH-terminal amino acid sequence of the hybrid protein from pTUBE638, it was shown that in , the NH-terminally extended thermostable α-amylase was translocated and remained in the periplasm after the 31 amino acid signal sequence was removed. In the case of , after the removal of a 34-amino acid signal sequence, the hybrid protein was secreted and processed to the mature form.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-136-8-1551
1990-08-01
2022-01-16
Loading full text...

Full text loading...

/deliver/fulltext/micro/136/8/mic-136-8-1551.html?itemId=/content/journal/micro/10.1099/00221287-136-8-1551&mimeType=html&fmt=ahah

References

  1. Birnboim H.C., Doly J. 1979; A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 7:1513–1523
    [Google Scholar]
  2. Blobel G., Dobberstein B. 1975; Transfer of proteins across membranes. Journal of Cell Biology 67:835–851
    [Google Scholar]
  3. Chan S.J., Weiss J., Konrad M., White T., Bahl C., Yu S.-D., Marks D., Steiner D.F. 1981; Biosynthesis and periplasmic segregation of human proinsulin in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 78:5401–5405
    [Google Scholar]
  4. Chang S., Cohen S.N. 1979; High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Molecular and General Genetics 168:111–115
    [Google Scholar]
  5. Davis B.D., Tai P.-C. 1980; The mechanism of protein secretion across membranes. Nature; London: 283433–438
    [Google Scholar]
  6. Dretzen G., Bellard M., Sassone-Corsi P., Chambon P. 1981; A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Analytical Biochemistry 112:295–298
    [Google Scholar]
  7. Fuller R.S., Sterne R.E., Thorner J. 1988; Enzymes required for yeast prohormone processing. Annual Review of Physiology 50:345–362
    [Google Scholar]
  8. Fuwa H. 1954; A new method for microdetermination of amylase activity by the use of amylose as the substrate. Journal of Biochemistry 41:583–603
    [Google Scholar]
  9. Ikemura H., Inouye M. 1988; In vitro processing of prosubtilisin produced in Escherichia coli. Journal of Biological Chemistry 263:12959–12963
    [Google Scholar]
  10. Ikemura H., Takagi H., Inouye M. 1987; Requirement of prosequence for the production of active subtilisin E in Escherichia coli. Journal of Biological Chemistry 262:7859–7864
    [Google Scholar]
  11. Lacks S.A., Springhorn S.S. 1980; Renaturation of enzymes after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Journal of Biological Chemistry 255:7467–7473
    [Google Scholar]
  12. Laemmli U.K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature; London: 227680–685
    [Google Scholar]
  13. Lederberg E.M., Cohen S.N. 1974; Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. Journal of Bacteriology 119:1072–1074
    [Google Scholar]
  14. Mäntsälä P. 1979; Membrane-bound and soluble extracellular α-amylase from Bacillus subtilis. Journal of Biological Chemistry 254:8540–8547
    [Google Scholar]
  15. Neurath H., Walsh K.A. 1976; Role of proteolytic enzymes in biological regulation. Proceedings of the National Academy of Sciences of the United States of America 73:3825–3832
    [Google Scholar]
  16. Nielsen J.B.K., Lampen J.O. 1982; Membrane-bound penicillinase in gram-positive bacteria. Journal of Biological Chemistry 257:4490–4495
    [Google Scholar]
  17. Sanger F., Nicklen S., Coulson A.R. 1977; DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences of the United States of America 74:5463–5467
    [Google Scholar]
  18. Sasamoto H., Nakazawa K., Tsutsumi K., Takase K., Yamane K. 1989; Signal peptide of Bacillus subtilis α-amylase. Journal of Biochemistry 106:376–382
    [Google Scholar]
  19. Shortle D. 1983; A genetic system for analysis of staphylococcal nuclease. Gene 22:181–189
    [Google Scholar]
  20. Sohma A., Fujita T., Yamane K. 1987; Protein processing to form extracellular thermostable α-amylases from a gene fused in a Bacillus subtilis secretion vector. Journal of General Microbiology 133:3271–3277
    [Google Scholar]
  21. Steiner D.F., Quinn P.S., Chan S.J., Marsh J., Tager H.S. 1980; Processing mechanisms in the biosynthesis of proteins. Annals of the New York Academy of Sciences 343:1–16
    [Google Scholar]
  22. Suominen I., Karp M., Lautamo J., Knowles J., Mantsala P. 1987; Thermostable α-amylase of Bacillus stearothermophilus: cloning, expression, and secretion by Escherichia coli. In Extracellular Enzymes of Microorganisms, pp 129–137 Chaloupka J., Krumphanzl V. Edited by New York: Plenum Press;
    [Google Scholar]
  23. Tabak H.F., Flavell R.A. 1978; A method for the recovery of DNA from agarose gels. Nucleic Acids Research 5:2321–2332
    [Google Scholar]
  24. Takase K., Mizuno H., Yamane K. 1988; NH2-terminal processing of Bacillus subtilis α-amylase. Journal of Biological Chemistry 263:11548–11553
    [Google Scholar]
  25. Vandekerckhove J., Bauw G., Puype M., Vandame J., VanMontagu M. 1985; Protein-blotting on polybrene-coated glass-fiber sheets. European Journal of Biochemistry 152:9–19
    [Google Scholar]
  26. Von Heijne G., Abrahmsen L. 1989; Species-specific variation in signal peptide design: implications for protein secretion in foreign hosts. FEBS Letters 244:439–446
    [Google Scholar]
  27. Watson M.E.E. 1984; Compilation of published signal sequences. Nucleic Acids Research 12:5145–5164
    [Google Scholar]
  28. Wong S.-L., Doi R.H. 1986; Determination of the signal peptidase cleavage site in the preprosubtilisin of Bacillus subtilis. Journal of Biological Chemistry 261:10176–10181
    [Google Scholar]
  29. Yamane K., Takeichi Y., Masuda T., Kawamura F., Saito H. 1982; Construction and physical map of a Bacillus subtilisspecialized transducing phage p 11 containing Bacillus subtilislys+ gene. Journal of General and Applied Microbiology 28:417–428
    [Google Scholar]
  30. Yamazaki H., Ohmura K., Nakayama A., Takeichi Y., Otozai K., Yamasaki M., Tamura G., Yamane K. 1983; α-amylase genes (amyR2 and amyE) from an α-amylase-hyperproducingBacillus subtilis strain: molecular cloning and nucleotide sequences. Journal of Bacteriology 156:327–337
    [Google Scholar]
  31. Yang M.Y., Ferrari E., Henner D.J. 1984; Cloning of the neutral protease gene of Bacillus subtilis and the use of the cloned gene to create an in vitro-derived deletion mutation. Journal of Bacteriology 160:15–21
    [Google Scholar]
  32. Yanisch-Perron C., Vieira J., Messing J. 1985; Improved M13 phage cloning vectors and host strains: nucleotide sequences of Ml 3 mpl8 and pUC19 vectors. Gene 33:103–119
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-136-8-1551
Loading
/content/journal/micro/10.1099/00221287-136-8-1551
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error