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Expression and purification of the SsbB protein from Streptococcus pneumoniae.

Authors
Hedayati-MA; Grove-DE; Steffen-SE; Bryant-FR
Source
Protein Expr Purif 2005 Oct; 43(2):133-139
NIOSHTIC No.
20038112
Abstract
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.
Keywords
Genes; Bacteria; Proteins; Author Keywords: Streptococcus pneumoniae; SSB protein; Single-stranded DNA binding protein; Natural transformation; SsbB protein
Contact
Floyd R. Bryant, Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, MD 21205
CODEN
PEXPEJ
Publication Date
20051001
Document Type
Journal Article
Email Address
fbryant@jhsph.edu
Funding Type
Grant
Fiscal Year
2006
NTIS Accession No.
NTIS Price
Identifying No.
Grant-Number-T42-OH-008428
Issue of Publication
2
ISSN
1046-5928
Source Name
Protein Expression and Purification
State
MD
Performing Organization
Johns Hopkins University
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