Structural and functional characterisation of recombinant human haemoglobin A expressed in Saccharomyces cerevisiae.

Recombinant human HbA, produced by co-expressing alpha-globin and beta-globin chains in the yeast Saccharomyces cerevisiae, has been characterised extensively both physically and functionally. Structural studies using N-terminal sequence analysis, peptide mapping, amino acid composition analysis and electrospray MS demonstrated that the recombinant protein was identical to standard HbA purified from erythrocytes. The functional properties of the recombinant protein were assessed using equilibrium and kinetic measurements of oxygen and carbon monoxide binding. The oxygen-binding studies demonstrated that the yeast-derived HbA behaved as a fully functional, cooperative tetramer (Hill coefficient, 2.9), exhibited a normal Bohr effect and response to phosphate, and displayed a rate of oxygen dissociation identical to that of the native human molecule. The recombinant protein also showed the same characteristics of carbon monoxide combination as the standard protein. These studies demonstrate that yeast provides an ideal system for the production of Hb for structural and functional analysis and a potentially useful source of HbA for formulation into a Hb-based oxygen carrier.

Production of recombinant human serum albumin from Saccharomyces cerevisiae.

Human serum albumin has been constitutively expressed in a Saccharomyces cerevisiae brewing yeast. After cell growth and disruption the product was associated with the insoluble fraction and represented approximately 1% of total cell protein. After the cell debris was extensively washed, the albumin was solubilized with 8 M urea and 28 mM 2-mercaptoethanol in 50 mM sodium carbonate buffer, pH 10. The denatured albumin was refolded by dialysis and further purified by anion exchange and gel filtration chromatography. Losses of renatured material could be reduced, or higher protein concentrations used during refolding, if the denatured product was purified by cation-exchange chromatography in urea prior to refolding. Apart from an additional N-terminal N-acetyl methionine, the refolded product proved identical to human serum albumin derived from plasma when compared by a variety of physical, chemical, and biological analytical methods.

Isolation and preliminary taxonomic studies of Thermus strains isolated from Yellowstone National Park, USA.

Forty-eight strains of Thermus isolated from hot springs in Yellowstone National Park, Wyoming, USA, and eight reference strains were subjected to a numerical taxonomic analysis using Gower's coefficient (SG) with single and average linkage clustering. Two major groups were distinguished, which could be differentiated by colony morphology, ability to reduce nitrate and proteolytic activity. Cluster 1 contained Thermus aquaticus YT-1, the type strain of the species, and cluster 2 contained authentic strains of 'T. flavus' and 'T. thermophilus'. T. ruber was recovered as a single member cluster. The mol % G + C of DNA from representative strains from each cluster was 64.4 to 66.8 for cluster 1, 62.2 to 67.1 for cluster 2 and 62.5 for T. ruber.

Investigation of cadmium resistance in an Alcaligenes sp.

The mechanisms of metal resistance of a cadmium-resistant Alcaligenes sp. were studied. Growth in a defined medium was unaffected by cadmium at concentrations up to 0.1 mM, while at concentrations up to 2.5 mM, growth occurred after an extended lag phase. The increase in length of the lag phase was abolished by repeated subculturing at these higher concentrations. However, subculture in the absence of cadmium reversed the adaptation process. Plasmid DNA was not detected in adapted cells, suggesting that adaptation is not plasmid mediated. Increased sulfide production in response to cadmium was observed, although the levels were too low to account fully for cadmium resistance. Adaptation of cells to cadmium resulted in the appearance of a major new membrane protein (molecular weight, 34,500) whose presence was not dependent upon the method of membrane preparation. This protein was induced at cadmium concentrations of 0.1 mM and above, but below this level the protein was absent. The onset of growth at concentrations above 0.1 mM was coincident with the appearance of this protein, which was also induced by zinc (0.4 mM) but not by manganese or nickel. The protein was only solubilized by a sodium dodecyl sulfate-2-mercaptoethanol mixture. Similar solubility properties were shown by a second major membrane protein (molecular weight, 33,000). These two proteins proved to be similar by peptide-mapping experiments and amino acid analysis. The appearance of the 34,500-molecular-weight protein and its possible role in cadmium resistance are discussed.