Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1984 Oct;81(19):5956–5960. doi: 10.1073/pnas.81.19.5956

Synthesis of a gene for human growth hormone and its expression in Escherichia coli.

M Ikehara, E Ohtsuka, T Tokunaga, Y Taniyama, S Iwai, K Kitano, S Miyamoto, T Ohgi, Y Sakuragawa, K Fujiyama, et al.
PMCID: PMC391837  PMID: 6091124

Abstract

A gene coding for human growth hormone, which consists of 192 amino acids, was chemically synthesized. The synthesis entailed ligating 78 deoxyribooligonucleotides, which had been synthesized on polymer supports by the phosphotriester method with frequently occurring amino acid codons of Escherichia coli. The chemically synthesized gene was inserted into an E. coli plasmid downstream from the E. coli trp promoter, with a modified ribosome-binding region carried on pBR322. E. coli cells transformed with this recombinant plasmid synthesized 2.9 X 10(6) molecules per cell of human growth hormone upon induction. The induced polypeptide was identical with natural human growth hormone in size and in immunological properties, as well as in biological activity as examined by the tibial test with hypophysectomized rats.

Full text

PDF
5960

Images in this article

5959Image
on p.5959
5960Image
on p.5960

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Broka C., Hozumi T., Arentzen R., Itakura K. Simplications in the synthesis of short oligonucleotide blocks. Nucleic Acids Res. 1980 Nov 25;8(22):5461–5471. doi: 10.1093/nar/8.22.5461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Crea R., Kraszewski A., Hirose T., Itakura K. Chemical synthesis of genes for human insulin. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5765–5769. doi: 10.1073/pnas.75.12.5765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Edge M. D., Green A. R., Heathcliffe G. R., Meacock P. A., Schuch W., Scanlon D. B., Atkinson T. C., Newton C. R., Markham A. F. Total synthesis of a human leukocyte interferon gene. Nature. 1981 Aug 20;292(5825):756–762. doi: 10.1038/292756a0. [DOI] [PubMed] [Google Scholar]
  4. Edge M. D., Greene A. R., Heathcliffe G. R., Moore V. E., Faulkner N. J., Camble R., Petter N. N., Trueman P., Schuch W., Hennam J. Chemical synthesis of a human interferon-alpha 2 gene and its expression in Escherichia coli. Nucleic Acids Res. 1983 Sep 24;11(18):6419–6435. doi: 10.1093/nar/11.18.6419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fritz H. J., Belagaje R., Brown E. L., Fritz R. H., Jones R. A., Lees R. G., Khorana H. G. High-pressure liquid chromatography in polynucleotide synthesis. Biochemistry. 1978 Apr 4;17(7):1257–1267. doi: 10.1021/bi00600a020. [DOI] [PubMed] [Google Scholar]
  6. GREENSPAN F. S., LI C. H. Bioassay of hypophyseal growth hormone; the tibia test. Endocrinology. 1949 Nov;45(5):455-63, illust. doi: 10.1210/endo-45-5-455. [DOI] [PubMed] [Google Scholar]
  7. Goeddel D. V., Heyneker H. L., Hozumi T., Arentzen R., Itakura K., Yansura D. G., Ross M. J., Miozzari G., Crea R., Seeburg P. H. Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone. Nature. 1979 Oct 18;281(5732):544–548. doi: 10.1038/281544a0. [DOI] [PubMed] [Google Scholar]
  8. Grantham R., Gautier C., Gouy M. Codon frequencies in 119 individual genes confirm consistent choices of degenerate bases according to genome type. Nucleic Acids Res. 1980 May 10;8(9):1893–1912. doi: 10.1093/nar/8.9.1893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hager D. A., Burgess R. R. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem. 1980 Nov 15;109(1):76–86. doi: 10.1016/0003-2697(80)90013-5. [DOI] [PubMed] [Google Scholar]
  10. Hallewell R. A., Emtage S. Plasmid vectors containing the tryptophan operon promoter suitable for efficient regulated expression of foreign genes. Gene. 1980 Apr;9(1-2):27–47. doi: 10.1016/0378-1119(80)90165-1. [DOI] [PubMed] [Google Scholar]
  11. Hsiung H. M., Sung W. L., Brousseau R., Wu R., Narang S. A. Synthesis of the human insulin gene. Part III. Chemical synthesis of 5'-phosphomonoester group containing deoxyribooligonucleotides by the modified phosphotriester method. Its application in the synthesis of seventeen fragments constituting human insulin C-chain DNA. Nucleic Acids Res. 1980 Dec 11;8(23):5753–5765. doi: 10.1093/nar/8.23.5753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ikemura T. Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system. J Mol Biol. 1981 Sep 25;151(3):389–409. doi: 10.1016/0022-2836(81)90003-6. [DOI] [PubMed] [Google Scholar]
  13. Itakura K., Hirose T., Crea R., Riggs A. D., Heyneker H. L., Bolivar F., Boyer H. W. Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science. 1977 Dec 9;198(4321):1056–1063. doi: 10.1126/science.412251. [DOI] [PubMed] [Google Scholar]
  14. Ito H., Ike Y., Ikuta S., Itakura K. Solid phase synthesis of polynucleotides. VI. Further studies on polystyrene copolymers for the solid support. Nucleic Acids Res. 1982 Mar 11;10(5):1755–1769. doi: 10.1093/nar/10.5.1755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Khorana H. G., Agarwal K. L., Büchi H., Caruthers M. H., Gupta N. K., Kleppe K., Kumar A., Otsuka E., RajBhandary U. L., Van de Sande J. H. Studies on polynucleotides. 103. Total synthesis of the structural gene for an alanine transfer ribonucleic acid from yeast. J Mol Biol. 1972 Dec 28;72(2):209–217. doi: 10.1016/0022-2836(72)90146-5. [DOI] [PubMed] [Google Scholar]
  16. Khorana H. G. Total synthesis of a gene. Science. 1979 Feb 16;203(4381):614–625. doi: 10.1126/science.366749. [DOI] [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Martial J. A., Hallewell R. A., Baxter J. D., Goodman H. M. Human growth hormone: complementary DNA cloning and expression in bacteria. Science. 1979 Aug 10;205(4406):602–607. doi: 10.1126/science.377496. [DOI] [PubMed] [Google Scholar]
  19. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  20. Ohtsuka E., Fujiyama K., Ikehara M. Studies on transfer ribonucleic acids and related compounds. XL. Synthesis of an eicosaribonucleotide corresponding to residues 35-54 of tRNAfMet from E. coli. Nucleic Acids Res. 1981 Jul 24;9(14):3503–3522. doi: 10.1093/nar/9.14.3503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ohtsuka E., Taniyama Y., Marumoto R., Sato H., Hirosaki H., Ikehara M. Studies on deoxyribonucleic acids and related compounds. V. Synthesis of pentadecanucleotide duplex containing the ideal Pribnow sequence of promoter by the phosphotriester method using a new phosphorylating reagent. Nucleic Acids Res. 1982 Apr 24;10(8):2597–2608. doi: 10.1093/nar/10.8.2597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sadler J. R., Tecklenburg M., Betz J. L. Plasmids containing many tandem copies of a synthetic lactose operator. Gene. 1980 Feb;8(3):279–300. doi: 10.1016/0378-1119(80)90005-0. [DOI] [PubMed] [Google Scholar]
  23. Sanger F., Donelson J. E., Coulson A. R., Kössel H., Fischer D. Use of DNA polymerase I primed by a synthetic oligonucleotide to determine a nucleotide sequence in phage fl DNA. Proc Natl Acad Sci U S A. 1973 Apr;70(4):1209–1213. doi: 10.1073/pnas.70.4.1209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Suzuki M., Sumi S., Hasegawa A., Nishizawa T., Miyoshi K., Wakisaka S., Miyake T., Misoka F. Production in Escherichia coli of biologically active secretin, a gastrointestinal hormone. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2475–2479. doi: 10.1073/pnas.79.8.2475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tanaka S., Oshima T., Ohsuye K., Ono T., Mizono A., Ueno A., Nakazato H., Tsujimoto M., Higashi N., Noguchi T. Expression in Escherichia coli of chemically synthesized gene for the human immune interferon. Nucleic Acids Res. 1983 Mar 25;11(6):1707–1723. doi: 10.1093/nar/11.6.1707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tanaka T., Letsinger R. L. Syringe method for stepwise chemical synthesis of oligonucleotides. Nucleic Acids Res. 1982 May 25;10(10):3249–3260. doi: 10.1093/nar/10.10.3249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Yanofsky C., Platt T., Crawford I. P., Nichols B. P., Christie G. E., Horowitz H., VanCleemput M., Wu A. M. The complete nucleotide sequence of the tryptophan operon of Escherichia coli. Nucleic Acids Res. 1981 Dec 21;9(24):6647–6668. doi: 10.1093/nar/9.24.6647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Yokota T., Sugisaki H., Takanami M., Kaziro Y. The nucleotide sequence of the cloned tufA gene of Escherichia coli. Gene. 1980 Dec;12(1-2):25–31. doi: 10.1016/0378-1119(80)90012-8. [DOI] [PubMed] [Google Scholar]
  29. de Boer H. A., Comstock L. J., Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983 Jan;80(1):21–25. doi: 10.1073/pnas.80.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES