Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1990 Oct 25;18(20):6075–6081. doi: 10.1093/nar/18.20.6075

Synthesis of a gene for human serum albumin and its expression in Saccharomyces cerevisiae.

M Kálmán 1, I Cserpán 1, G Bajszár 1, A Dobi 1, E Horváth 1, C Pázmán 1, A Simoncsits 1
PMCID: PMC332408  PMID: 2235491

Abstract

A 1761 base pairs long artificial gene coding for human serum albumin (HSA) has been prepared by a newly developed synthetic approach, resulting in the largest synthetic gene so far described. Oligonucleotides corresponding to only one strand of the HSA gene were prepared by chemical synthesis, while the complementary strand was obtained by a combination of enzymatic and cloning steps. 24 synthetic, 69-85 nucleotides long oligonucleotides covering the major part of the HSA gene (41-1761 nucleotides) were used as building blocks. Generally, four groups of 6-6 such oligonucleotides were successively cloned in pUC19 Escherichia coli vector to obtain about quarters of the gene as large fragments. Joining of these four fragments resulted in a cloned DNA coding for the 13-585 amino acid region of HSA, which was further supplemented with a double-stranded linker sequence coding for the amino terminal 12 amino acids. The completed structural gene composed of frequently used codons in the highly expressed yeast genes was then supplied with yeast regulatory sequences and the HSA expression cassette so obtained was inserted into an Escherichia coli-Saccharomyces cerevisiae shuttle vector. This vector was shown to direct the expression in Saccharomyces cerevisiae of correctly processed, mature HSA which was recognized by antiserum to HSA, and possessed the correct N-terminal amino acid sequence.

Full text

PDF
6075

Images in this article

6078Image
on p.6078
6079Image
on p.6079
6079Image
on p.6079

Selected References

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

  1. Adams S. E., Johnson I. D., Braddock M., Kingsman A. J., Kingsman S. M., Edwards R. M. Synthesis of a gene for the HIV transactivator protein TAT by a novel single stranded approach involving in vivo gap repair. Nucleic Acids Res. 1988 May 25;16(10):4287–4298. doi: 10.1093/nar/16.10.4287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bajwa W., Meyhack B., Rudolph H., Schweingruber A. M., Hinnen A. Structural analysis of the two tandemly repeated acid phosphatase genes in yeast. Nucleic Acids Res. 1984 Oct 25;12(20):7721–7739. doi: 10.1093/nar/12.20.7721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beggs J. D. Transformation of yeast by a replicating hybrid plasmid. Nature. 1978 Sep 14;275(5676):104–109. doi: 10.1038/275104a0. [DOI] [PubMed] [Google Scholar]
  4. Bell L. D., Smith J. C., Derbyshire R., Finlay M., Johnson I., Gilbert R., Slocombe P., Cook E., Richards H., Clissold P. Chemical synthesis, cloning and expression in mammalian cells of a gene coding for human tissue-type plasminogen activator. Gene. 1988 Mar 31;63(2):155–163. doi: 10.1016/0378-1119(88)90521-5. [DOI] [PubMed] [Google Scholar]
  5. Bennetzen J. L., Hall B. D. Codon selection in yeast. J Biol Chem. 1982 Mar 25;257(6):3026–3031. [PubMed] [Google Scholar]
  6. Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
  7. Chen H. B., Weng J. M., Jiang K., Bao J. S. A new method for the synthesis of a structural gene. Nucleic Acids Res. 1990 Feb 25;18(4):871–878. doi: 10.1093/nar/18.4.871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clark J. M., Joyce C. M., Beardsley G. P. Novel blunt-end addition reactions catalyzed by DNA polymerase I of Escherichia coli. J Mol Biol. 1987 Nov 5;198(1):123–127. doi: 10.1016/0022-2836(87)90462-1. [DOI] [PubMed] [Google Scholar]
  9. Clark J. M. Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 1988 Oct 25;16(20):9677–9686. doi: 10.1093/nar/16.20.9677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cousens D. J., Wilson M. J., Hinchliffe E. Construction of a regulated PGK expression vector. Nucleic Acids Res. 1990 Mar 11;18(5):1308–1308. doi: 10.1093/nar/18.5.1308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Denèfle P., Kovarik S., Guitton J. D., Cartwright T., Mayaux J. F. Chemical synthesis of a gene coding for human angiogenin, its expression in Escherichia coli and conversion of the product into its active form. Gene. 1987;56(1):61–70. doi: 10.1016/0378-1119(87)90158-2. [DOI] [PubMed] [Google Scholar]
  12. Derbyshire K. M., Salvo J. J., Grindley N. D. A simple and efficient procedure for saturation mutagenesis using mixed oligodeoxynucleotides. Gene. 1986;46(2-3):145–152. doi: 10.1016/0378-1119(86)90398-7. [DOI] [PubMed] [Google Scholar]
  13. Dugaiczyk A., Law S. W., Dennison O. E. Nucleotide sequence and the encoded amino acids of human serum albumin mRNA. Proc Natl Acad Sci U S A. 1982 Jan;79(1):71–75. doi: 10.1073/pnas.79.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Ferretti L., Karnik S. S., Khorana H. G., Nassal M., Oprian D. D. Total synthesis of a gene for bovine rhodopsin. Proc Natl Acad Sci U S A. 1986 Feb;83(3):599–603. doi: 10.1073/pnas.83.3.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Geli V., Baty D., Knibiehler M., Lloubès R., Pessegue B., Shire D., Lazdunski C. Synthesis and sequence-specific proteolysis of a hybrid protein (colicin A::growth hormone releasing factor) produced in Escherichia coli. Gene. 1989 Aug 1;80(1):129–136. doi: 10.1016/0378-1119(89)90257-6. [DOI] [PubMed] [Google Scholar]
  17. Gröger G., Ramalho-Ortigao F., Steil H., Seliger H. A comprehensive list of chemically synthesized genes. Nucleic Acids Res. 1988 Aug 25;16(16):7763–7771. doi: 10.1093/nar/16.16.7763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hostomský Z., Smrt J., Arnold L., Tocík Z., Paces V. Solid-phase assembly of cow colostrum trypsin inhibitor gene. Nucleic Acids Res. 1987 Jun 25;15(12):4849–4856. doi: 10.1093/nar/15.12.4849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Howell M. L., Blumenthal K. M. Cloning and expression of a synthetic gene for Cerebratulus lacteus neurotoxin B-IV. J Biol Chem. 1989 Sep 15;264(26):15268–15273. [PubMed] [Google Scholar]
  20. 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]
  21. Jay E., MacKnight D., Lutze-Wallace C., Harrison D., Wishart P., Liu W. Y., Asundi V., Pomeroy-Cloney L., Rommens J., Eglington L. Chemical synthesis of a biologically active gene for human immune interferon-gamma. Prospect for site-specific mutagenesis and structure-function studies. J Biol Chem. 1984 May 25;259(10):6311–6317. [PubMed] [Google Scholar]
  22. 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]
  23. Lawn R. M., Adelman J., Bock S. C., Franke A. E., Houck C. M., Najarian R. C., Seeburg P. H., Wion K. L. The sequence of human serum albumin cDNA and its expression in E. coli. Nucleic Acids Res. 1981 Nov 25;9(22):6103–6114. doi: 10.1093/nar/9.22.6103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mandecki W., Bolling T. J. FokI method of gene synthesis. Gene. 1988 Aug 15;68(1):101–107. doi: 10.1016/0378-1119(88)90603-8. [DOI] [PubMed] [Google Scholar]
  25. Mazin A. V., Saparbaev M. K., Ovchinnikova L. P., Dianov G. L., Salganik R. I. Site-directed insertion of long single-stranded DNA fragments into plasmid DNA. DNA Cell Biol. 1990 Jan-Feb;9(1):63–69. doi: 10.1089/dna.1990.9.63. [DOI] [PubMed] [Google Scholar]
  26. Meloun B., Morávek L., Kostka V. Complete amino acid sequence of human serum albumin. FEBS Lett. 1975 Oct 15;58(1):134–137. doi: 10.1016/0014-5793(75)80242-0. [DOI] [PubMed] [Google Scholar]
  27. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nassal M. Total chemical synthesis of a gene for hepatitis B virus core protein and its functional characterization. Gene. 1988 Jun 30;66(2):279–294. doi: 10.1016/0378-1119(88)90364-2. [DOI] [PubMed] [Google Scholar]
  29. Papanicolaou C., Ripley L. S. Polymerase-specific differences in the DNA intermediates of frameshift mutagenesis. In vitro synthesis errors of Escherichia coli DNA polymerase I and its large fragment derivative. J Mol Biol. 1989 May 20;207(2):335–353. doi: 10.1016/0022-2836(89)90258-1. [DOI] [PubMed] [Google Scholar]
  30. Peters T., Jr Serum albumin. Adv Protein Chem. 1985;37:161–245. doi: 10.1016/s0065-3233(08)60065-0. [DOI] [PubMed] [Google Scholar]
  31. Rink H., Liersch M., Sieber P., Meyer F. A large fragment approach to DNA synthesis: total synthesis of a gene for the protease inhibitor eglin c from the leech Hirudo medicinalis and its expression in E. coli. Nucleic Acids Res. 1984 Aug 24;12(16):6369–6387. doi: 10.1093/nar/12.16.6369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rossi J. J., Kierzek R., Huang T., Walker P. A., Itakura K. An alternate method for synthesis of double-stranded DNA segments. J Biol Chem. 1982 Aug 25;257(16):9226–9229. [PubMed] [Google Scholar]
  33. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Saunders C. W., Schmidt B. J., Mallonee R. L., Guyer M. S. Secretion of human serum albumin from Bacillus subtilis. J Bacteriol. 1987 Jul;169(7):2917–2925. doi: 10.1128/jb.169.7.2917-2925.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sharp P. M., Tuohy T. M., Mosurski K. R. Codon usage in yeast: cluster analysis clearly differentiates highly and lowly expressed genes. Nucleic Acids Res. 1986 Jul 11;14(13):5125–5143. doi: 10.1093/nar/14.13.5125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sijmons P. C., Dekker B. M., Schrammeijer B., Verwoerd T. C., van den Elzen P. J., Hoekema A. Production of correctly processed human serum albumin in transgenic plants. Biotechnology (N Y) 1990 Mar;8(3):217–221. doi: 10.1038/nbt0390-217. [DOI] [PubMed] [Google Scholar]
  37. Sleep D., Belfield G. P., Goodey A. R. The secretion of human serum albumin from the yeast Saccharomyces cerevisiae using five different leader sequences. Biotechnology (N Y) 1990 Jan;8(1):42–46. doi: 10.1038/nbt0190-42. [DOI] [PubMed] [Google Scholar]
  38. Sproat B. S., Gait M. J. Chemical synthesis of a gene for somatomedin C. Nucleic Acids Res. 1985 Apr 25;13(8):2959–2977. doi: 10.1093/nar/13.8.2959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Storms R. K., McNeil J. B., Khandekar P. S., An G., Parker J., Friesen J. D. Chimeric plasmids for cloning of deoxyribonucleic acid sequences in Saccharomyces cerevisiae. J Bacteriol. 1979 Oct;140(1):73–82. doi: 10.1128/jb.140.1.73-82.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Struhl K. Nucleotide sequence and transcriptional mapping of the yeast pet56-his3-ded1 gene region. Nucleic Acids Res. 1985 Dec 9;13(23):8587–8601. doi: 10.1093/nar/13.23.8587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Uhlmann E. An alternative approach in gene synthesis: use of long selfpriming oligodeoxynucleotides for the construction of double-stranded DNA. Gene. 1988 Nov 15;71(1):29–40. doi: 10.1016/0378-1119(88)90074-1. [DOI] [PubMed] [Google Scholar]
  42. Vasser M., Ng P. G., Jhurani P., Bischofberger N. Error rates in oligodeoxynucleotides synthesized by the H-phosphonate method. Nucleic Acids Res. 1990 May 25;18(10):3089–3089. doi: 10.1093/nar/18.10.3089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Weiner M. P., Scheraga H. A. A method for the cloning of unpurified single-stranded oligonucleotides. Nucleic Acids Res. 1989 Sep 12;17(17):7113–7113. doi: 10.1093/nar/17.17.7113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wells J. A., Vasser M., Powers D. B. Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites. Gene. 1985;34(2-3):315–323. doi: 10.1016/0378-1119(85)90140-4. [DOI] [PubMed] [Google Scholar]
  45. Wosnick M. A., Barnett R. W., Carlson J. E. Total chemical synthesis and expression in Escherichia coli of a maize glutathione-transferase (GST) gene. Gene. 1989 Mar 15;76(1):153–160. doi: 10.1016/0378-1119(89)90017-6. [DOI] [PubMed] [Google Scholar]
  46. Wosnick M. A., Barnett R. W., Vicentini A. M., Erfle H., Elliott R., Sumner-Smith M., Mantei N., Davies R. W. Rapid construction of large synthetic genes: total chemical synthesis of two different versions of the bovine prochymosin gene. Gene. 1987;60(1):115–127. doi: 10.1016/0378-1119(87)90219-8. [DOI] [PubMed] [Google Scholar]
  47. Wychowski C., Emerson S. U., Silver J., Feinstone S. M. Construction of recombinant DNA molecules by the use of a single stranded DNA generated by the polymerase chain reaction: its application to chimeric hepatitis A virus/poliovirus subgenomic cDNA. Nucleic Acids Res. 1990 Feb 25;18(4):913–918. doi: 10.1093/nar/18.4.913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES