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. 1998 Mar 1;330(Pt 2):983–988. doi: 10.1042/bj3300983

Bacterial expression and spectroscopic characterization of soybean leghaemoglobin a.

D K Jones 1, R Badii 1, F I Rosell 1, E Lloyd 1
PMCID: PMC1219234  PMID: 9480919

Abstract

A gene encoding leghaemoglobin a from soybean has been constructed and the soluble recombinant protein expressed in E. coli. The integrity of the recombinant protein has been assessed by a range of spectroscopic techniques. Electrospray mass spectrometry of the protein indicates that the molecular mass of the protein corresponds to the predicted amino acid sequence. Circular dichroism spectra of the ferric derivative and UV-visible spectra of various ferric and ferrous derivatives (pH 6.99, mu = 0.10 M, 25.0 degrees C) are consistent with published data for the wild-type protein. For the ferric derivative, UV-visible (298 and 77 K) and EPR (10 K) spectra indicate the existence of a thermal equilibrium between high- and low-spin forms. Titration of the protein (0.10 M NaCl, mu = 0.10 M, 25.0 degrees C) between pHs 6.68 and 10.35 indicate formation (pKa = 8.3+/-0.03) of a 6-coordinate, hydroxide-bound form of the protein at high pH. All of the above data are consistent with the behaviour of the wild-type protein.

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Selected References

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  1. Appleby C. A., Blumberg W. E., Peisach J., Wittenberg B. A., Wittenberg J. B. Leghemoglobin. An electron paramagnetic resonance and optical spectral study of the free protein and its complexes with nicotinate and acetate. J Biol Chem. 1976 Oct 10;251(19):6090–6096. [PubMed] [Google Scholar]
  2. Appleby C. A., Nicola N. A., Hurrell J. G., Leach S. J. Characterization and improved separation of soybean leghemoglobins. Biochemistry. 1975 Oct 7;14(20):4444–4450. doi: 10.1021/bi00691a016. [DOI] [PubMed] [Google Scholar]
  3. Arredondo-Peter R., Moran J. F., Sarath G., Luan P., Klucas R. V. Molecular cloning of the cowpea leghemoglobin II gene and expression of its cDNA in Escherichia coli. Purification and characterization of the recombinant protein. Plant Physiol. 1997 Jun;114(2):493–500. doi: 10.1104/pp.114.2.493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Badii R., Basran J., Casarotto M. G., Roberts G. C. High-level expression and isotopic labeling of Lactobacillus casei dihydrofolate reductase for nuclear magnetic resonance spectroscopy. Protein Expr Purif. 1995 Jun;6(3):237–243. doi: 10.1006/prep.1995.1030. [DOI] [PubMed] [Google Scholar]
  5. Davidowitz E. J., Creissen G., Vincze E., Kiss G. B., Lang-Unnasch N. Sequence analysis of alfalfa (Medicago sativa) leghemoglobin cDNA and genomic clones. Plant Mol Biol. 1991 Jan;16(1):161–165. doi: 10.1007/BF00017926. [DOI] [PubMed] [Google Scholar]
  6. Davidowitz E. J., Dow A., Lang-Unnasch N. Nucleotide sequence of a cDNA clone encoding a leghemoglobin from Medicago sativa. Nucleic Acids Res. 1989 Apr 25;17(8):3307–3307. doi: 10.1093/nar/17.8.3307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Edwards G. M., Huber H. E., DeFeo-Jones D., Vuocolo G., Goodhart P. J., Maigetter R. Z., Sanyal G., Oliff A., Heimbrook D. C. Purification and characterization of a functionally homogeneous 60-kDa species of the retinoblastoma gene product. J Biol Chem. 1992 Apr 25;267(12):7971–7974. [PubMed] [Google Scholar]
  9. Ellfolk N., Sievers G. Circular dichroism of soybean leghemoglobin. Biochim Biophys Acta. 1975 Oct 20;405(2):213–227. doi: 10.1016/0005-2795(75)90088-4. [DOI] [PubMed] [Google Scholar]
  10. Ellfolk N., Sievers G. Correction of the amino acid sequence of soybean leghemoglobin alpha. Acta Chem Scand B. 1974;28(10):1245–1246. doi: 10.3891/acta.chem.scand.28b-1245. [DOI] [PubMed] [Google Scholar]
  11. Ellfolk N., Sievers G. The primary structure of soybean leghemoglobin. Acta Chem Scand. 1971;25(9):3532–3534. doi: 10.3891/acta.chem.scand.25-3532. [DOI] [PubMed] [Google Scholar]
  12. Ellis P. J., Appleby C. A., Guss J. M., Hunter W. N., Ollis D. L., Freeman H. C. Structure of ferric soybean leghemoglobin a nicotinate at 2.3 A resolution. Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):302–310. doi: 10.1107/S0907444997000292. [DOI] [PubMed] [Google Scholar]
  13. Fuchsman W. H., Appleby C. A. CO and O2 complexes of soybean leghemoglobins: pH effects upon infrared and visible spectra. Comparisons with CO and O2 complexes of myoglobin and hemoglobin. Biochemistry. 1979 Apr 3;18(7):1309–1321. doi: 10.1021/bi00574a030. [DOI] [PubMed] [Google Scholar]
  14. Hargrove M. S., Barry J. K., Brucker E. A., Berry M. B., Phillips G. N., Jr, Olson J. S., Arredondo-Peter R., Dean J. M., Klucas R. V., Sarath G. Characterization of recombinant soybean leghemoglobin a and apolar distal histidine mutants. J Mol Biol. 1997 Mar 14;266(5):1032–1042. doi: 10.1006/jmbi.1996.0833. [DOI] [PubMed] [Google Scholar]
  15. Hildebrand D. P., Burk D. L., Maurus R., Ferrer J. C., Brayer G. D., Mauk A. G. The proximal ligand variant His93Tyr of horse heart myoglobin. Biochemistry. 1995 Feb 14;34(6):1997–2005. doi: 10.1021/bi00006a021. [DOI] [PubMed] [Google Scholar]
  16. Hyldig-Nielsen J. J., Jensen E. O., Paludan K., Wiborg O., Garrett R., Jørgensen P., Marcker K. A. The primary structures of two leghemoglobin genes from soybean. Nucleic Acids Res. 1982 Jan 22;10(2):689–701. doi: 10.1093/nar/10.2.689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kiss G. B., Végh Z., Vincze E. Nucleotide sequence of a cDNA clone encoding leghemoglobin III (LbIII) from Medicago sativa. Nucleic Acids Res. 1987 Apr 24;15(8):3620–3620. doi: 10.1093/nar/15.8.3620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Konieczny A., Jensen E. O., Marcker K. A., Legocki A. B. Molecular cloning of lupin leghemoglobin cDNA. Mol Biol Rep. 1987;12(1):61–66. doi: 10.1007/BF00580652. [DOI] [PubMed] [Google Scholar]
  19. Konieczny A. Nucleotide sequence of lupin leghemoglobin I cDNA. Nucleic Acids Res. 1987 Aug 25;15(16):6742–6742. doi: 10.1093/nar/15.16.6742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lehtovaara P. Studies on ligand binding of kidney bean leghemoglobin. Acta Chem Scand B. 1977;31(1):21–27. doi: 10.3891/acta.chem.scand.31b-0021. [DOI] [PubMed] [Google Scholar]
  21. Lloyd E., Burk D. L., Ferrer J. C., Maurus R., Doran J., Carey P. R., Brayer G. D., Mauk A. G. Electrostatic modification of the active site of myoglobin: characterization of the proximal Ser92Asp variant. Biochemistry. 1996 Sep 10;35(36):11901–11912. doi: 10.1021/bi9608976. [DOI] [PubMed] [Google Scholar]
  22. Löbler M., Hirsch A. M. An alfalfa (Medicago sativa L.) cDNA encoding an acidic leghemoglobin (MsLb3). Plant Mol Biol. 1992 Nov;20(4):733–736. doi: 10.1007/BF00046457. [DOI] [PubMed] [Google Scholar]
  23. Maskall C. S., Gibson J. F., Dart P. J. Electron-paramagnetic-resonance studies of leghaemoglobins from soya-bean and cowpea root nodules. Identification of nitrosyl-leghaemoglobin in crude leghaemoglobin preparations. Biochem J. 1977 Nov 1;167(2):435–445. doi: 10.1042/bj1670435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Prytulla S., Dyson H. J., Wright P. E. Gene synthesis, high-level expression and assignment of backbone 15N and 13C resonances of soybean leghemoglobin. FEBS Lett. 1996 Dec 16;399(3):283–289. doi: 10.1016/s0014-5793(96)01278-1. [DOI] [PubMed] [Google Scholar]
  25. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  26. Welters P., Metz B. A., Schell J., de Bruijn F. J. Nucleotide sequence of the Sesbania rostrata leghemoglobin (Srglb3) gene. Nucleic Acids Res. 1989 Feb 11;17(3):1253–1253. doi: 10.1093/nar/17.3.1253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wiborg O., Hyldig-Nielsen J. J., Jensen E. O., Paludan K., Marcker K. A. The nucleotide sequences of two leghemoglobin genes from soybean. Nucleic Acids Res. 1982 Jun 11;10(11):3487–3494. doi: 10.1093/nar/10.11.3487. [DOI] [PMC free article] [PubMed] [Google Scholar]

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