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. 1993 Sep 1;90(17):8108–8112. doi: 10.1073/pnas.90.17.8108

Production of unmodified human adult hemoglobin in Escherichia coli.

T J Shen 1, N T Ho 1, V Simplaceanu 1, M Zou 1, B N Green 1, M F Tam 1, C Ho 1
PMCID: PMC47297  PMID: 8367471

Abstract

We have constructed a plasmid (pHE2) in which the synthetic human alpha- and beta-globin genes and the methionine aminopeptidase (Met-AP) gene from Escherichia coli are coexpressed under the control of separate tac promoters. The Hbs were expressed in E. coli JM109 and purified by fast protein liquid chromatography, producing two major components, a and b. Electrospray mass spectrometry shows that at least 98% and about 90% of the expressed alpha and beta chains of component a, respectively, have the expected masses. The remaining 10% of the beta chain in component a corresponds in mass to the beta chain plus methionine. In component b, both alpha and beta chains have the correct masses without detectable N-terminal methionine (< 2%). These results have been confirmed by Edman degradation studies of the amino-terminal sequences of the alpha and beta chains of these two recombinant Hb (rHb) samples. rHbs from components a and b exhibit visible optical spectra identical to that of human normal adult Hb (Hb A). Component a and Hb A have very similar oxygen-binding properties, but component b shows somewhat altered oxygen binding, especially at low pH values. 1H-NMR spectra of component a and Hb A are essentially identical, whereas those of component b exhibit altered ring current-shifted and hyperfine-shifted proton resonances, indicating altered heme conformation in the beta chain. These altered resonance patterns can be changed to those of Hb A by converting component b to the ferric state and then to the deoxy state and finally back to either the carbonmonoxy or oxy form. Thus, our E. coli expression system produces native, unmodified Hb A in high yield and can be used to produce desired mutant Hbs.

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Selected References

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

  1. Behringer R. R., Ryan T. M., Reilly M. P., Asakura T., Palmiter R. D., Brinster R. L., Townes T. M. Synthesis of functional human hemoglobin in transgenic mice. Science. 1989 Sep 1;245(4921):971–973. doi: 10.1126/science.2772649. [DOI] [PubMed] [Google Scholar]
  2. Ben-Bassat A., Bauer K., Chang S. Y., Myambo K., Boosman A., Chang S. Processing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure. J Bacteriol. 1987 Feb;169(2):751–757. doi: 10.1128/jb.169.2.751-757.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bucci E. Preparation of isolated chains of human hemoglobin. Methods Enzymol. 1981;76:97–106. doi: 10.1016/0076-6879(81)76117-2. [DOI] [PubMed] [Google Scholar]
  4. Chada K., Magram J., Raphael K., Radice G., Lacy E., Costantini F. Specific expression of a foreign beta-globin gene in erythroid cells of transgenic mice. 1985 Mar 28-Apr 3Nature. 314(6009):377–380. doi: 10.1038/314377a0. [DOI] [PubMed] [Google Scholar]
  5. Chang Y. H., Teichert U., Smith J. A. Molecular cloning, sequencing, deletion, and overexpression of a methionine aminopeptidase gene from Saccharomyces cerevisiae. J Biol Chem. 1992 Apr 25;267(12):8007–8011. [PubMed] [Google Scholar]
  6. Chang Y. H., Teichert U., Smith J. A. Purification and characterization of a methionine aminopeptidase from Saccharomyces cerevisiae. J Biol Chem. 1990 Nov 15;265(32):19892–19897. [PubMed] [Google Scholar]
  7. Fung L. W., Ho C. A proton nuclear magnetic resonance study of the quaternary structure of human homoglobins in water. Biochemistry. 1975 Jun 3;14(11):2526–2535. doi: 10.1021/bi00682a036. [DOI] [PubMed] [Google Scholar]
  8. Groebe D. R., Busch M. R., Tsao T. Y., Luh F. Y., Tam M. F., Chung A. E., Gaskell M., Liebhaber S. A., Ho C. High-level production of human alpha- and beta-globins in insect cells. Protein Expr Purif. 1992 Apr;3(2):134–141. doi: 10.1016/s1046-5928(05)80097-x. [DOI] [PubMed] [Google Scholar]
  9. Hayashi A., Suzuki T., Shin M. An enzymic reduction system for metmyoglobin and methemoglobin, and its application to functional studies of oxygen carriers. Biochim Biophys Acta. 1973 Jun 15;310(2):309–316. doi: 10.1016/0005-2795(73)90110-4. [DOI] [PubMed] [Google Scholar]
  10. Hernan R. A., Hui H. L., Andracki M. E., Noble R. W., Sligar S. G., Walder J. A., Walder R. Y. Human hemoglobin expression in Escherichia coli: importance of optimal codon usage. Biochemistry. 1992 Sep 15;31(36):8619–8628. doi: 10.1021/bi00151a032. [DOI] [PubMed] [Google Scholar]
  11. Hewick R. M., Hunkapiller M. W., Hood L. E., Dreyer W. J. A gas-liquid solid phase peptide and protein sequenator. J Biol Chem. 1981 Aug 10;256(15):7990–7997. [PubMed] [Google Scholar]
  12. Hirel P. H., Schmitter M. J., Dessen P., Fayat G., Blanquet S. Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8247–8251. doi: 10.1073/pnas.86.21.8247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ho C. Proton nuclear magnetic resonance studies on hemoglobin: cooperative interactions and partially ligated intermediates. Adv Protein Chem. 1992;43:153–312. doi: 10.1016/s0065-3233(08)60555-0. [DOI] [PubMed] [Google Scholar]
  14. Hoffman S. J., Looker D. L., Roehrich J. M., Cozart P. E., Durfee S. L., Tedesco J. L., Stetler G. L. Expression of fully functional tetrameric human hemoglobin in Escherichia coli. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8521–8525. doi: 10.1073/pnas.87.21.8521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ishimori K., Morishima I. Study of the specific heme orientation in reconstituted hemoglobins. Biochemistry. 1988 Jun 28;27(13):4747–4753. doi: 10.1021/bi00413a025. [DOI] [PubMed] [Google Scholar]
  16. Kavanaugh J. S., Rogers P. H., Arnone A. High-resolution X-ray study of deoxy recombinant human hemoglobins synthesized from beta-globins having mutated amino termini. Biochemistry. 1992 Sep 15;31(36):8640–8647. doi: 10.1021/bi00151a034. [DOI] [PubMed] [Google Scholar]
  17. Kendall R. L., Bradshaw R. A. Isolation and characterization of the methionine aminopeptidase from porcine liver responsible for the co-translational processing of proteins. J Biol Chem. 1992 Oct 15;267(29):20667–20673. [PubMed] [Google Scholar]
  18. La Mar G. N., Nagai K., Jue T., Budd D. L., Gersonde K., Sick H., Kagimoto T., Hayashi A., Taketa F. Assignment of proximal histidyl imidazole exchangeable proton NMR resonances to individual subunits in hemoglobins A, Boston, Iwate and Milwaukee. Biochem Biophys Res Commun. 1980 Oct 16;96(3):1172–1177. doi: 10.1016/0006-291x(80)90075-3. [DOI] [PubMed] [Google Scholar]
  19. La Mar G. N., Yamamoto Y., Jue T., Smith K. M., Pandey R. K. 1H NMR characterization of metastable and equilibrium heme orientational heterogeneity in reconstituted and native human hemoglobin. Biochemistry. 1985 Jul 16;24(15):3826–3831. doi: 10.1021/bi00336a002. [DOI] [PubMed] [Google Scholar]
  20. Liddington R., Derewenda Z., Dodson G., Harris D. Structure of the liganded T state of haemoglobin identifies the origin of cooperative oxygen binding. Nature. 1988 Feb 25;331(6158):725–728. doi: 10.1038/331725a0. [DOI] [PubMed] [Google Scholar]
  21. Martin de Llano J. J., Schneewind O., Stetler G., Manning J. M. Recombinant human sickle hemoglobin expressed in yeast. Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):918–922. doi: 10.1073/pnas.90.3.918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Miller C. G., Strauch K. L., Kukral A. M., Miller J. L., Wingfield P. T., Mazzei G. J., Werlen R. C., Graber P., Movva N. R. N-terminal methionine-specific peptidase in Salmonella typhimurium. Proc Natl Acad Sci U S A. 1987 May;84(9):2718–2722. doi: 10.1073/pnas.84.9.2718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Movva N. R., Semon D., Meyer C., Kawashima E., Wingfield P., Miller J. L., Miller C. G. Cloning and nucleotide sequence of the Salmonella typhimurium pepM gene. Mol Gen Genet. 1990 Sep;223(2):345–348. doi: 10.1007/BF00265075. [DOI] [PubMed] [Google Scholar]
  24. Nagai K., Thøgersen H. C. Generation of beta-globin by sequence-specific proteolysis of a hybrid protein produced in Escherichia coli. 1984 Jun 28-Jul 4Nature. 309(5971):810–812. doi: 10.1038/309810a0. [DOI] [PubMed] [Google Scholar]
  25. Nagai K., Thøgersen H. C. Synthesis and sequence-specific proteolysis of hybrid proteins produced in Escherichia coli. Methods Enzymol. 1987;153:461–481. doi: 10.1016/0076-6879(87)53072-5. [DOI] [PubMed] [Google Scholar]
  26. Nakamura K., Nakamura A., Takamatsu H., Yoshikawa H., Yamane K. Cloning and characterization of a Bacillus subtilis gene homologous to E. coli secY. J Biochem. 1990 Apr;107(4):603–607. doi: 10.1093/oxfordjournals.jbchem.a123093. [DOI] [PubMed] [Google Scholar]
  27. ROSSI-FANELLI A., ANTONINI E., CAPUTO A. Studies on the structure of hemoglobin. II. Properties of reconstituted protohemoglobin and protoporphyrin-globin. Biochim Biophys Acta. 1959 Sep;35:93–101. doi: 10.1016/0006-3002(59)90338-5. [DOI] [PubMed] [Google Scholar]
  28. Ryan T. M., Behringer R. R., Townes T. M., Palmiter R. D., Brinster R. L. High-level erythroid expression of human alpha-globin genes in transgenic mice. Proc Natl Acad Sci U S A. 1989 Jan;86(1):37–41. doi: 10.1073/pnas.86.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Swanson M. E., Martin M. J., O'Donnell J. K., Hoover K., Lago W., Huntress V., Parsons C. T., Pinkert C. A., Pilder S., Logan J. S. Production of functional human hemoglobin in transgenic swine. Biotechnology (N Y) 1992 May;10(5):557–559. doi: 10.1038/nbt0592-557. [DOI] [PubMed] [Google Scholar]
  30. Takahashi S., Lin A. K., Ho C. Proton nuclear magnetic resonance studies of hemoglobins M Boston (alpha 58E7 His leads to Tyr) and M Milwaukee (beta 67E11 Val leads to Glu): spectral assignments of hyperfine-shifted proton resonances and of proximal histidine (E7) NH resonances to the alpha and beta chains of normal human adult hemoglobin. Biochemistry. 1980 Nov 11;19(23):5196–5202. doi: 10.1021/bi00564a007. [DOI] [PubMed] [Google Scholar]
  31. Townes T. M., Lingrel J. B., Chen H. Y., Brinster R. L., Palmiter R. D. Erythroid-specific expression of human beta-globin genes in transgenic mice. EMBO J. 1985 Jul;4(7):1715–1723. doi: 10.1002/j.1460-2075.1985.tb03841.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tsunasawa S., Stewart J. W., Sherman F. Amino-terminal processing of mutant forms of yeast iso-1-cytochrome c. The specificities of methionine aminopeptidase and acetyltransferase. J Biol Chem. 1985 May 10;260(9):5382–5391. [PubMed] [Google Scholar]
  33. Wagenbach M., O'Rourke K., Vitez L., Wieczorek A., Hoffman S., Durfee S., Tedesco J., Stetler G. Synthesis of wild type and mutant human hemoglobins in Saccharomyces cerevisiae. Biotechnology (N Y) 1991 Jan;9(1):57–61. doi: 10.1038/nbt0191-57. [DOI] [PubMed] [Google Scholar]
  34. Waks M., Yip Y. K., Beychok S. Influence of prosthetic groups on protein folding and subunit assembly. Recombination of separated human alpha-and beta-globin chains with heme and alloplex interactions of globin chains with heme-containing subunits. J Biol Chem. 1973 Sep 25;248(18):6462–6470. [PubMed] [Google Scholar]
  35. Yamamoto Y., La Mar G. N. 1H NMR study of dynamics and thermodynamics of heme rotational disorder in native and reconstituted hemoglobin A. Biochemistry. 1986 Sep 9;25(18):5288–5297. doi: 10.1021/bi00366a045. [DOI] [PubMed] [Google Scholar]

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