Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.332009
Title: Studies on the expression of a cloned Staphylococcus aureus protein A gene in Escherichia coli
Author: Shuttleworth, Helen L.
ISNI:       0000 0001 3408 2313
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1991
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Abstract:
The gene coding for staphylococcal protein A has been cloned and the nucleotide sequence coding for the structural gene and its 5' flanking region determined. The DNA sequence showed the gene to be composed of a series of repetitive sequences, with five 120 nucleotide repeats comprising the 5' part of the structural gene and ten 24 nucleotide repeats in a continuous sequence at the 3’ terminus. Analysis of codon usage of the gene and comparison with other S. aureus genes revealed that spa showed a higher degree of homology with S. aureus plasmid genes than with chromosomally- located genes. Comparison with the reported codon preference in E.coli showed spa to have a similar bias, except for the leu codon. An open reading frame of the spa structural gene showed 508 a.a residues, with a predicted protein molecular weight of 55,426. The N-terminal signal peptide comprised the first 36 amino acids. A fifth N-terminal domain (E) was shown to be homologous to the four IgG-binding domains previously reported to constitute the N-terminal portion of SpA. The C-terminal part of SpA was shown to consist of two structurally different regions: an N-terminal repetitive region (Xr) composed of ten octapeptide repeats which are hydrophilic and have been proposed to span the Gram-positive cell wall, followed by a single domain (Xc), 58 a.a in length, whose C-terminal portion contains 20 hydrophobic residues, suggesting anchorage on the cell membrane. Enhanced production of SpA in E. coli has been achieved by placing the E. coli lac promoter immediately upstream of the spa gene. This has allowed yields as high as 1.5g of SpA per litre of culture to be obtained from 150-litre batch fermenters. Problems were encountered during large-scale fermentation, as the recombinant clones were found to be extremely fragile when producing high levels of SpA. Characterisation of the recombinant SpA indicated a molecular weight of 54,000 for the full length protein, compared with 56,000 for SpA produced in S. aureus. SDS-PAGE analysis of the IgG-binding proteins revealed extensive proteolysis of the recombinant SpA. Comparitive analysis of the IgG-binding polypeptides encoded by a truncated spa gene (lacking 69 a.a residues from the C-terminus), together with the determination of the N-terminal amino acid sequence, indicated that proteolysis occurred at the N- terminal portion of the Xc region. Cell fractionation studies and immunoelectronmicroscopy localised the SpA to the periplasm in E. coli. A possible regulatory control region for the spa gene has been localised upstream of the structural gene. The E.coli alkaline phosphatase gene was isolated, cloned and its sequence determined to facilitate the production of SpA-alkaline phosphatase fusion proteins.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.332009  DOI: Not available
Keywords: QR Microbiology
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