Compaction agent clarification of microbial lysates

Brad W. DeWalt, Jason C. Murphy, George E. Fox, Richard C. Willson

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

Recombinant proteins are often purified from microbial lysates containing high concentrations of nucleic acids. Pre-purification steps such as nuclease addition or precipitation with polyethyleneimine or ammonium sulfate are normally required to reduce viscosity and to eliminate competing polyanions before anion exchange chromatography. We report that small polycationic compaction agents such as spermine selectively precipitate nucleic acids during or after Escherichia coli lysis, allowing DNA and RNA to be pelleted with the insoluble cell debris. Analysis by spectrophotometry and protein assay confirmed a significant reduction in the concentration of nucleic acids present, with preservation of protein. Lysate viscosity is greatly reduced, facilitating subsequent processing. We have used 5 mM spermine to remove nucleic acids from E. coli lysate in the purification of a hexahistidine-tagged HIV reverse transcriptase.

Original languageEnglish (US)
Pages (from-to)220-223
Number of pages4
JournalProtein Expression and Purification
Volume28
Issue number2
DOIs
StatePublished - Apr 1 2003

ASJC Scopus subject areas

  • Biotechnology

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