Compaction agent clarification of microbial lysates

Autor:	Brad W. DeWalt, Richard C. Willson, Jason C. Murphy, George E. Fox
Rok vydání:	2003
Předmět:	DNA Bacterial Nuclease Lysis Recombinant Fusion Proteins Spermine RNA Biology medicine.disease_cause RNA Bacterial chemistry.chemical_compound Bacteriolysis chemistry Biochemistry Escherichia coli Nucleic acid biology.protein medicine Isoelectric Focusing Bradford protein assay DNA Biotechnology
Zdroj:	Protein Expression and Purification. 28:220-223
ISSN:	1046-5928
DOI:	10.1016/s1046-5928(02)00677-0
Popis:	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 5mM spermine to remove nucleic acids from E. coli lysate in the purification of a hexahistidine-tagged HIV reverse transcriptase.
Databáze:	OpenAIRE
Externí odkaz:	https://explore.openaire.eu/search/publication?articleId=doi_dedup___::6aaf2068fefecc6caf352039aacecc88 https://doi.org/10.1016/s1046-5928(02)00677-0 Zobrazit plný text záznamu Full Text from ScienceDirect