A High-Throughput Strategy for Recombinant Protein Expression and Solubility Screen in Escherichia coli : A Case of Sensor Histidine Kinase

  • Agnieszka Szmitkowska
  • Blanka Pekárová
  • Jan HejátkoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2077)


Determining conditions optimal for host growth, maximal protein yield, and lysis buffer composition is of critical importance for the efficient purification of soluble and well-folded recombinant proteins suitable for functional and/or structural studies. Small-scale optimization of conditions for protein production and stability saves time, labor, and costs. Here we describe a protocol for quick protein production and solubility screen using TissueLyser II system from Qiagen enabling simultaneous processing of 96 protein samples, with application to recombinant proteins encompassing two intracellular domains of ethylene-recognizing sensor histidine kinase ETHYLENE RESPONSE1 (ETR1) from Arabidopsis thaliana. We demonstrate that conditions for expression and cell lysis found in our small-scale screen allow successful large-scale production of pure and functional domains of sensor histidine kinase, providing a strategy potentially transferable to other similar catalytic domains.

Key words

Protein Expression screen Solubility screen Growth conditions Histidine kinase Sample preparation 



The work was supported by the Ministry of Education, Youth and Sports of the Czech Republic under the projects CEITEC 2020 (LQ1601).


  1. 1.
    Structural Genomics C, China Structural Genomics C, Northeast Structural Genomics C et al (2008) Protein production and purification. Nat Methods 5:135–146CrossRefGoogle Scholar
  2. 2.
    Mizianty MJ, Kurgan L (2011) Sequence-based prediction of protein crystallization, purification and production propensity. Bioinformatics 27:i24–i33CrossRefGoogle Scholar
  3. 3.
    Vincentelli R, Canaan S, Offant J et al (2005) Automated expression and solubility screening of His-tagged proteins in 96-well format. Anal Biochem 346:77–84CrossRefGoogle Scholar
  4. 4.
    Braun P, Labaer J (2003) High throughput protein production for functional proteomics. Trends Biotechnol 21:383–388CrossRefGoogle Scholar
  5. 5.
    Berrow NS, Bussow K, Coutard B et al (2006) Recombinant protein expression and solubility screening in Escherichia coli: a comparative study. Acta Crystallogr D Biol Crystallogr 62:1218–1226CrossRefGoogle Scholar
  6. 6.
    Vincentelli R, Cimino A, Geerlof A et al (2011) High-throughput protein expression screening and purification in Escherichia coli. Methods 55:65–72CrossRefGoogle Scholar
  7. 7.
    Correa A, Ortega C, Obal G et al (2014) Generation of a vector suite for protein solubility screening. Front Microbiol 5:67CrossRefGoogle Scholar
  8. 8.
    Bjerga GE, Arsin H, Larsen O et al (2016) A rapid solubility-optimized screening procedure for recombinant subtilisins in E. coli. J Biotechnol 222:38–46CrossRefGoogle Scholar
  9. 9.
    Listwan P, Terwilliger TC, Waldo GS (2009) Automated, high-throughput platform for protein solubility screening using a split-GFP system. J Struct Funct Genom 10:47–55CrossRefGoogle Scholar
  10. 10.
    Cold Spring Harb Protoc. 2006. doi:
  11. 11.
  12. 12.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  13. 13.
  14. 14.
    Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354CrossRefGoogle Scholar
  15. 15.
    Ueno TB, Johnson RA, Boon EM (2015) Optimized assay for the quantification of histidine kinase autophosphorylation. Biochem Biophys Res Commun 465:331–337CrossRefGoogle Scholar
  16. 16.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  17. 17.
    Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:172PubMedPubMedCentralGoogle Scholar
  18. 18.
    Gamble RL, Coonfield ML, Schaller GE (1998) Histidine kinase activity of the ETR1 ethylene receptor from Arabidopsis. Proc Natl Acad Sci U S A 95:7825–7829CrossRefGoogle Scholar
  19. 19.
    Moussatche P, Klee HJ (2004) Autophosphorylation activity of the Arabidopsis ethylene receptor multigene family. J Biol Chem 279:48734–48741CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Agnieszka Szmitkowska
    • 1
  • Blanka Pekárová
    • 1
  • Jan Hejátko
    • 1
    Email author
  1. 1.Central European Institute of Technology and National Centre for Biomolecular Research, Faculty of ScienceMasaryk UniversityBrnoCzech Republic

Personalised recommendations