Selective release of overexpressed recombinant proteins from E. coli cells facilitates one-step chromatographic purification of peptide-tagged green fluorescent protein variants

Yasmin Kaveh-Baghbaderani, Silvia Angela Blank-Shim, Tobias Koch, Sonja Berensmeier

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

The need for fast and convenient protein purification is enormous, not only on the industrial scale for the production of therapeutics but also in life science research. Most protein purification strategies require multiple steps, which are time consuming, cost and resource intensive. Selective release of the intracellular target product can considerably facilitate downstream processing by minimizing the HCP concentration before subsequent purification processes. Here, we present a simple method to selectively release soluble overexpressed proteins from E. coli with minimal laboratory equipment requirements. This method is based on the creation of pores in the cell envelope by a single freeze-thaw cycle. Proteins of various sizes were released by several 50 mM Tris pH 9.5 buffer washes with high yields. Consequently, the need for detergents and other additives were circumvented. Green fluorescent protein (GFP), was tagged with peptides of various charges and polarity. Releasing these GFP variants with our method enabled the successful one-step chromatographic purification on a quaternary amine resin despite the different isoelectric points of the target proteins. Up to 90% of the target protein elution peak during anion exchange chromatography was of >95% purity. This optimized process is more productive compared to multi-step purification procedures currently available in literature.

Original languageEnglish
Pages (from-to)155-160
Number of pages6
JournalProtein Expression and Purification
Volume152
DOIs
StatePublished - Dec 2018

Keywords

  • Freeze-thaw
  • Green fluorescent protein GFP
  • One-step
  • Protein purification
  • Selective liberation
  • Selective release

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