Mutational analysis of sickle haemoglobin (Hb) gelation Journal Article
| Authors: | Li, X.; Himanen, J. P.; Martin De Llano, J. J.; Padovan, J. C.; Chait, B. T.; Manning, J. M. |
| Article Title: | Mutational analysis of sickle haemoglobin (Hb) gelation |
| Abstract: | The use of recombinant Hb has provided the advantage that any amino acid substitution can be made at sites not represented by natural mutants or that cannot be modified by chemical procedures. We have recently reported the expression of human sickle Hb (HbS) in the yeast Saccharomyces cerevisiae that carries a plasmid containing the human α- and β-globin cDNA sequences; N-terminal nascent protein processing is correct and a soluble correctly folded Hb tetramer is produced. The yeast system produces a recombinant sickle Hb that is identical by about a dozen biochemical and physiological criteria with the natural sickle Hb purified from the red cells of sickle-cell anaemia patients. Most importantly, the gelling concentration of this recombinant sickle Hb is the same as that of the HbS purified from human sickle red cells. The misfolding of Hb reported for the Escherichia coli-expressed protein is not apparent for Hb expressed in yeast by any of the criteria that we have used for characterization. These findings indicate that this system is well suited to the production of HbS mutants to explore those areas of the HbS tetramer whose roles in the gelation process are not yet defined and to measure quantitatively the strength of such interactions at certain inter-tetrameric contact sites in the deoxy-HbS aggregate. This article reviews our studies on a number of sickle Hb mutants, including polymerization-enhancing HbS mutants and polymerization-inhibiting HbS mutants. |
| Keywords: | human cell; mass spectrometry; amino acid substitution; amino acid sequence; saccharomyces cerevisiae; polymerization; recombinant proteins; recombinant protein; plasmid; base sequence; dna sequence; mutagenesis, site-directed; chromatography, high pressure liquid; dna primers; amino acids; site directed mutagenesis; complementary dna; dna, complementary; circular dichroism; sickle cell anemia; isoelectric focusing; hemoglobin s; humans; human; article; biopolymers; hemoglobin, sickle |
| Journal Title: | Biotechnology and Applied Biochemistry |
| Volume: | 29 |
| Issue: | 2 |
| ISSN: | 0885-4513 |
| Publisher: | John Wiley & Sons |
| Date Published: | 1999-04-01 |
| Start Page: | 165 |
| End Page: | 184 |
| Language: | English |
| PUBMED: | 10075913 |
| PROVIDER: | scopus |
| DOI: | 10.1111/j.1470-8744.1999.tb00546.x |
| DOI/URL: | |
| Notes: | Article -- Export Date: 16 August 2016 -- Source: Scopus |
