Expression and purification of the SsbB protein from Streptococcus pneumoniae

Details

• Personal Author:
  Bryant FR ; Grove DE ; Hedayati MA ; Steffen SE
• Description:
  The Gram positive bacterium, Streptococcus pneumoniae, has two genes, designated ssbA and ssbB, which are predicted to encode single-stranded DNA binding proteins (SSB proteins). We have shown previously that the SsbA protein is similar in size and in biochemical properties to the well-characterized SSB protein from Escherichia coli. The SsbB protein, in contrast, is a smaller protein and has no counterpart in E. coli. This report describes the development of an expression system and purification procedure for the SsbB protein. The ssbB gene was amplified from genomic S. pneumoniae DNA and cloned into the E. coli expression vector, pET21a. Although, we had shown previously that the SsbA protein is strongly expressed from pET21a in the E. coli strain BL21(DE3)pLysS, no expression of the SsbB protein was detected in these cells. However, the SsbB protein was strongly expressed from pET21a in the Rosetta(DE3)pLysS strain, a derivative of BL21(DE3)pLysS which supplies the tRNAs for six codons that are used infrequently in E. coli. The differential expression of the two SSB proteins in the parent BL21(DE3)pLysS strain was apparently due to the presence of two rare codons in the ssbB gene sequence that are not present in the ssbA sequence. Using the Rosetta(DE3)pLysS/pETssbB expression system, a protocol was developed in which the SsbB protein was purified to apparent homogeneity. DNA binding assays confirmed that the purified SsbB protein had single-stranded DNA binding activity. The expression and purification procedures reported here will facilitate further investigations into the biological role of the SsbB protein. [Description provided by NIOSH]
• Subjects:
  Genetics Occupational Health Safety
• Keywords:
  Author Keywords: Streptococcus Pneumoniae Bacteria Genes Natural Transformation Proteins Single-stranded DNA Binding Protein SsbB Protein SSB Protein
• ISSN:
  1046-5928
• Document Type:
  Text
• Funding:
  Grant
• Genre:
  Journal Article
• Place as Subject:
  Maryland ; OSHA Region 3
• CIO:
  National Institute for Occupational Safety and Health (NIOSH)
• Topic:
  Workplace Safety & Health
• Location:
  North America
• Pages in Document:
  133-139
• Volume:
  43
• Issue:
  2
• NIOSHTIC Number:
  nn:20038112
• Citation:
  Protein Expr Purif 2005 Oct; 43(2):133-139
• Contact Point Address:
  Floyd R. Bryant, Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, MD 21205
• Email:
  fbryant@jhsph.edu
• Federal Fiscal Year:
  2006
• Performing Organization:
  Johns Hopkins University
• Peer Reviewed:
  True
• Start Date:
  20050701
• Source Full Name:
  Protein Expression and Purification
• End Date:
  20280630
• Collection(s):
  National Institute for Occupational Safety and Health
• Main Document Checksum:
  urn:sha-512:41bdedfa871c4121d419b466f6bb88aada004d51846685ecdb3e8e64c5296d347b58c860afd42fe9cf2a45a56bfd5c351e1dd75d79eafacf93cfb9e09f4902e5
• Download URL:
  https://stacks.cdc.gov/view/cdc/191922/cdc_191922_DS1.pdf
• File Type:
  [PDF - 270.26 KB ]