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. 1993 Nov 1;295(Pt 3):719–724. doi: 10.1042/bj2950719

Purification and biochemical characterization of recombinant human placental growth hormone produced in Escherichia coli.

A Igout 1, J Van Beeumen 1, F Frankenne 1, M L Scippo 1, B Devreese 1, G Hennen 1
PMCID: PMC1134619  PMID: 8240283

Abstract

The hGH-V (or hGH-2) gene codes for human placental growth hormone (hPGH). Secretion of hPGH is continuous, in contrast with the pulsed secretion of pituitary growth hormone (hGH) which it progressively replaces in the maternal bloodstream. hGH-V cDNA has previously been cloned and isolated. Analysis of its nucleotide sequence has revealed a 191-residue protein, hPGH, differing from hGH at 13 positions. The calculated pI is more basic than that of the pituitary hormone. Here we have inserted hGH-V cDNA into the pIN-III-ompA3 plasmid in order to produce hPGH in its native form in Escherichia coli D1210. Expression of hGH-V cDNA in E. coli is significantly lower than that of hGH cDNA with the same expression system. The hPGH produced in E. coli was purified in quantities sufficient to allow its biochemical and immunochemical characterization. The molecular mass of the protein was determined by electrospray m.s. The determined mass, 22,320 Da, agrees well with the molecular mass calculated from the translated cDNA sequence, assuming the presence of two disulphide bridges. Having established the technique for producing hPGH with a primary structure identical to the natural, non-glycosylated, 22 kDa isoform, we can now plan the full physicochemical and pharmaceutical characterization of this new hormonal entity.

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Selected References

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

  1. Barsh G. S., Seeburg P. H., Gelinas R. E. The human growth hormone gene family: structure and evolution of the chromosomal locus. Nucleic Acids Res. 1983 Jun 25;11(12):3939–3958. doi: 10.1093/nar/11.12.3939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bolivar F., Backman K. Plasmids of Escherichia coli as cloning vectors. Methods Enzymol. 1979;68:245–267. doi: 10.1016/0076-6879(79)68018-7. [DOI] [PubMed] [Google Scholar]
  3. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Chen E. Y., Liao Y. C., Smith D. H., Barrera-Saldaña H. A., Gelinas R. E., Seeburg P. H. The human growth hormone locus: nucleotide sequence, biology, and evolution. Genomics. 1989 May;4(4):479–497. doi: 10.1016/0888-7543(89)90271-1. [DOI] [PubMed] [Google Scholar]
  6. Cooke N. E., Ray J., Emery J. G., Liebhaber S. A. Two distinct species of human growth hormone-variant mRNA in the human placenta predict the expression of novel growth hormone proteins. J Biol Chem. 1988 Jun 25;263(18):9001–9006. [PubMed] [Google Scholar]
  7. Eriksson L., Frankenne F., Edèn S., Hennen G., Von Schoultz B. Growth hormone 24-h serum profiles during pregnancy--lack of pulsatility for the secretion of the placental variant. Br J Obstet Gynaecol. 1989 Aug;96(8):949–953. doi: 10.1111/j.1471-0528.1989.tb03352.x. [DOI] [PubMed] [Google Scholar]
  8. Fenn J. B., Mann M., Meng C. K., Wong S. F., Whitehouse C. M. Electrospray ionization for mass spectrometry of large biomolecules. Science. 1989 Oct 6;246(4926):64–71. doi: 10.1126/science.2675315. [DOI] [PubMed] [Google Scholar]
  9. Frankenne F., Closset J., Gomez F., Scippo M. L., Smal J., Hennen G. The physiology of growth hormones (GHs) in pregnant women and partial characterization of the placental GH variant. J Clin Endocrinol Metab. 1988 Jun;66(6):1171–1180. doi: 10.1210/jcem-66-6-1171. [DOI] [PubMed] [Google Scholar]
  10. Frankenne F., Scippo M. L., Van Beeumen J., Igout A., Hennen G. Identification of placental human growth hormone as the growth hormone-V gene expression product. J Clin Endocrinol Metab. 1990 Jul;71(1):15–18. doi: 10.1210/jcem-71-1-15. [DOI] [PubMed] [Google Scholar]
  11. Ghrayeb J., Kimura H., Takahara M., Hsiung H., Masui Y., Inouye M. Secretion cloning vectors in Escherichia coli. EMBO J. 1984 Oct;3(10):2437–2442. doi: 10.1002/j.1460-2075.1984.tb02151.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gomez F., Pirens G., Schaus C., Closset J., Hennen G. A highly sensitive radioimmunoassay for human growth hormone using a monoclonal antibody. J Immunoassay. 1984;5(3-4):145–157. doi: 10.1080/01971528408063005. [DOI] [PubMed] [Google Scholar]
  13. Hennen G., Frankenne F., Closset J., Gomez F., Smal J., el Khayat N. Physiologie de l'hormone de croissance au cours de la grossesse et découverte d'une hormone de croissance placentaire humaine. Bull Mem Acad R Med Belg. 1986;141(1):111–119. [PubMed] [Google Scholar]
  14. Hennen G., Frankenne F., Pirens G., Gomez F., Closset J., Schaus C., el Khayat N. New chorionic GH-like antigen revealed by monoclonal antibody radioimmunoassays. Lancet. 1985 Feb 16;1(8425):399–399. doi: 10.1016/s0140-6736(85)91420-5. [DOI] [PubMed] [Google Scholar]
  15. Hirt H., Kimelman J., Birnbaum M. J., Chen E. Y., Seeburg P. H., Eberhardt N. L., Barta A. The human growth hormone gene locus: structure, evolution, and allelic variations. DNA. 1987 Feb;6(1):59–70. doi: 10.1089/dna.1987.6.59. [DOI] [PubMed] [Google Scholar]
  16. Igout A., Scippo M. L., Frankenne F., Hennen G. Cloning and nucleotide sequence of placental hGH-V cDNA. Arch Int Physiol Biochim. 1988 Mar;96(1):63–67. doi: 10.3109/13813458809079626. [DOI] [PubMed] [Google Scholar]
  17. Igout A., Scippo M. L., Frankenne F., Hennen G. Expression and secretion of the human placental growth hormone in Escherichia coli. Nucleic Acids Res. 1989 May 25;17(10):3998–3998. doi: 10.1093/nar/17.10.3998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Koshland D., Botstein D. Secretion of beta-lactamase requires the carboxy end of the protein. Cell. 1980 Jul;20(3):749–760. doi: 10.1016/0092-8674(80)90321-9. [DOI] [PubMed] [Google Scholar]
  19. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Osborn M. J., Gander J. E., Parisi E., Carson J. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J Biol Chem. 1972 Jun 25;247(12):3962–3972. [PubMed] [Google Scholar]
  22. Owerbach D., Rutter W. J., Martial J. A., Baxter J. D., Shows T. B. Genes for growth hormone, chorionic somatommammotropin, and growth hormones-like gene on chromosome 17 in humans. Science. 1980 Jul 11;209(4453):289–292. doi: 10.1126/science.7384802. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]

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