Large-Scale Overproduction and Rapid Purification of the Escherichia coli ssb Gene Product. Expression of the ssb Gene under λ PL Control

Timothy M. Lohman, J. Michael Green, Richard S. Beyer

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192 Scopus citations

Abstract

We report a rapid procedure for the large-scale purification of the Escherichia coli encoded single-strand binding (SSB) protein, a helix-destabilizing protein which is essential for replication, recombination, and repair processes in E. coli. To facilitate the isolation of large quantities of the ssb gene product, we have subcloned the ssb gene into a temperature-inducible expression vector, pPLc28 [Remaut, E., Stanssens, P., & Fiers, W. (1981) Gene 15, 81–93], carrying the bacteriophage λ PL promoter. A large overproduction of the ssb gene product results upon shifting the temperature of E. coli strains which carry the plasmid and also produce the thermolabile λcI857 repressor. After 5 h of induction, the ssb gene product represents ~ 10% of the total cell protein. The overexpression of the ssb gene and the purification protocol reported here enable one to isolate SSB protein (>99% pure) with final yields of ~3 mg of SSB protein/g of cell paste. In fact, very pure (>99%) SSB protein can be obtained after approximately 8 h, starting from frozen cells in the absence of any columns, although inclusion of a single-stranded DNA-cellulose column is generally recommended to ensure that the purified SSB protein possesses DNA binding activity. The ability to easily purify 1 g of SSB protein from 300–350 g of induced cells will facilitate physical studies requiring large quantities of this important protein.

Original languageEnglish
Pages (from-to)21-25
Number of pages5
JournalBiochemistry
Volume25
Issue number1
DOIs
StatePublished - Jan 1 1986

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