Correct assembly of human normal adult hemoglobin when expressed in transgenic swine: Chemical, conformational and functional equivalence with the human-derived protein

Belur N. Manjula, Ramesh Kumar, D. Philip Sun, Nancy T. Ho, Chien Ho, Janardhan M. Rao, Ashok Malavalli, A. Seetharama Acharya

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Structural and functional investigations of recombinant human hemoglobin A (HbA) isolated from the erythrocytes of transgenic swine coexpressing human α- and β-globins have been carried out to authenticate its correct expression, post-translational processing and assembly. The HbA expressed in transgenic swine (TgHbA) is indistinguishable from the human-derived HbA in terms of its isoelectric pH, mass and elution pattern on a Mono S column. The chemical identity of the α- and β-globin chains of TgHbA with the corresponding chains from human-derived HbA has been established by tryptic peptide mapping and amino acid sequencing. The proton NMR spectra of TgHbA have demonstrated that the conformational aspects of the protein around the heme pocket are indistinguishable from those of the control sample of HbA. The equivalence of the hydrogen bond pattern of TgHbA (in particular the inter-subunit surfaces) with that of authentic HbA has also been established by NMR studies. Consistent with these structural and conformational analyses, the TgHbA also exhibits complete functional equivalence with the human-derived HbA with respect to oxygen affinity, cooperativity, Bohr effect and allostery. Hence the studies presented here demonstrate that the transgenic swine system correctly transcribes the α- and β-globin transgenes, translates the respective α- and β-globin mRNA to generate the corresponding globin chains, carries out the correct cotranslational processing of the translated globin chains, inserts the heme into the globin chains in the same orientation as in the human-derived HbA and assembles the α- and β-subunits into a functionally cooperative tetramer that exhibits a response to allosteric effecters identical with that of human-derived HbA. Thus, in the transgenic swine system, in vitro chemical manipulation steps such as those needed in the Escherichia coli and the yeast systems, to convert the rHbA expressed in these systems into forms functionally identical with that of the human-derived protein, are not needed. An additional advantage of the transgenic swine system is the stability of the transgenes over many generations. Hence the transgenic swine could serve as an excellent system for the production of human HbA (or its variants) for structure-function studies and for therapeutic applications.

Original languageEnglish (US)
Pages (from-to)583-588
Number of pages6
JournalProtein Engineering
Volume11
Issue number7
StatePublished - Jul 1998

Fingerprint

Hemoglobin A
Hemoglobin
Globins
Hemoglobins
Swine
Proteins
Heme
Transgenes
Nuclear magnetic resonance
Peptide Mapping
Protein Sequence Analysis
Processing
Yeast
Escherichia coli
Peptides
Protons
Amino acids
Hydrogen
Hydrogen bonds
Yeasts

Keywords

  • Human normal adult hemoglobin assembly
  • Transgenic swine

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry

Cite this

Correct assembly of human normal adult hemoglobin when expressed in transgenic swine : Chemical, conformational and functional equivalence with the human-derived protein. / Manjula, Belur N.; Kumar, Ramesh; Sun, D. Philip; Ho, Nancy T.; Ho, Chien; Rao, Janardhan M.; Malavalli, Ashok; Acharya, A. Seetharama.

In: Protein Engineering, Vol. 11, No. 7, 07.1998, p. 583-588.

Research output: Contribution to journalArticle

Manjula, Belur N. ; Kumar, Ramesh ; Sun, D. Philip ; Ho, Nancy T. ; Ho, Chien ; Rao, Janardhan M. ; Malavalli, Ashok ; Acharya, A. Seetharama. / Correct assembly of human normal adult hemoglobin when expressed in transgenic swine : Chemical, conformational and functional equivalence with the human-derived protein. In: Protein Engineering. 1998 ; Vol. 11, No. 7. pp. 583-588.
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AU - Ho, Nancy T.

AU - Ho, Chien

AU - Rao, Janardhan M.

AU - Malavalli, Ashok

AU - Acharya, A. Seetharama

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