Increasing the soluble expression and crystallization of the Escherichia coli quorum-sensing protein LsrK

Jung Hye Ha, Yumi Eo, Hee Chul Ahn, Kyoung Seok Ryu

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

LsrK is one of the key components of the luxS-regulated (lsr) operon in Escherichia coli and plays an important role during the quorum-sensing (QS) process mediated by autoinducer-2 (AI-2). The AI-2 molecule is imported into the cell by the LsrACB transporter and is subsequently phosphorylated (to AI-2-P) by LsrK. AI-2-P binds to the repressor protein of the lsr operon (LsrR) and triggers various cellular responses related to QS by dissociating LsrR from the DNA. Although a large amount of purified LsrK is required for structural studies, recombinant GST-LsrK was mostly expressed in an insoluble form. To enhance the soluble expression of LsrK, an attempt was made to increase the expression of the cellular chaperone proteins that are well known to support proper protein folding. Transformed E. coli was cultured in high-salt LB medium and heat shock was applied prior to subsequent IPTG induction at 20C. These procedures increased the yield of purified LsrK by about tenfold compared with standard IPTG induction at 20C. The expressed LsrK was readily purified by GST-affinity chromatography. Crystals of LsrK were grown by the hanging-drop vapour-diffusion method. The X-ray diffraction data of the crystal were processed in a primitive hexagonal space group to 2.9Å resolution.LsrK from E. coli phosphorylates imported AI-2 molecules and triggers quorum-sensing signalling. The protein was expressed by applying heat and osmotic shock before IPTG induction, and a crystal of LsrK diffracted to 2.9Å resolution.

Original languageEnglish
Pages (from-to)253-258
Number of pages6
JournalActa Crystallographica Section F:Structural Biology Communications
Volume73
Issue number5
DOIs
StatePublished - May 2017

Keywords

  • chaperones
  • heat shock
  • LsrK complex
  • LsrK crystal
  • osmotic shock
  • quorum sensing
  • soluble expression of LsrK

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