Evaluation of Escherichia coli cell disruption and inclusion body release using nucleic acid binding fluorochromes and flow cytometry

Many types of commercially valuable recombinant proteins produced by fermentation are expressed at high levels in Escherichia coli. Often, high-level expression in the host results in the formation of insoluble inclusion bodies. The release of these intracellular inclusion bodies from E. coli follow...

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Bibliographic Details
Published inBioTechniques Vol. 43; no. 6; pp. 777 - 782
Main Authors MEDWID, Richard D, KREBS, Lara, WELCH, Shellie
Format Journal Article
LanguageEnglish
Published Natick, MA Eaton 01.12.2007
Taylor & Francis Group
Subjects
Biological and medical sciences
Diverse techniques
Escherichia coli - genetics
Escherichia coli - metabolism
Fermentation
Flow Cytometry - methods
Fluorescent Dyes
Fundamental and applied biological sciences. Psychology
Inclusion Bodies
Molecular and cellular biology
Molecular Biology - methods
Recombinant Proteins - genetics
Recombinant Proteins - isolation & purification
Solubility
Flow cytometry
Escherichia coli
Bacteria
Inclusion body
Nucleic acid
Release
Enterobacteriaceae
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Summary:Many types of commercially valuable recombinant proteins produced by fermentation are expressed at high levels in Escherichia coli. Often, high-level expression in the host results in the formation of insoluble inclusion bodies. The release of these intracellular inclusion bodies from E. coli following cell disruption is a requirement for further downstream recovery. The ability to discern between intact unruptured cells and granules released from broken cells can provide valuable information for improving recovery yields in downstream purification. This paper describes a rapid and sensitive cytometry-based method that allows the simultaneous measurement of intact heat-killed E. coli and inclusion bodies using staining with nucleic acid binding fluorochromes.
Bibliography:ObjectType-Article-2
SourceType-Scholarly Journals-1
ObjectType-Technical Report-1
ObjectType-Feature-3
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ISSN:0736-6205
1940-9818
DOI:10.2144/000112621