A novel flow cytometry-based method for analysis of expression levels in Escherichia coli, giving information about precipitated and soluble protein

J Biotechnol. 2005 Sep 23;119(2):133-46. doi: 10.1016/j.jbiotec.2005.03.024.

Abstract

A high throughput method for screening of protein expression is described. By using a flow cytometer, levels of both soluble and precipitated protein can simultaneously be assessed in vivo. Protein fragments were fused to the N-terminus of enhanced GFP and the cell samples were analysed using a flow cytometer. Data concerning whole cell fluorescence and light scattering was collected. The whole cell fluorescence is probing intracellular concentrations of soluble fusion proteins. Concurrently, forward scattered light gives data about inclusion body formation, valuable information in process optimisation. To evaluate the method, the cells were disrupted, separated into soluble and non-soluble fractions and analysed by gel electrophoresis. A clear correlation between fluorescence and soluble target protein was shown. Interestingly, the distribution of the cells regarding forward scatter (standard deviation) correlates with the amount of inclusion bodies formed. Finally, the newly developed method was used to evaluate two different purification tags, His(6) and Z(basic), and their effect on the expression pattern.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chemical Precipitation
  • Cloning, Molecular
  • Escherichia coli / cytology
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism*
  • Flow Cytometry / methods*
  • Gene Expression*
  • Inclusion Bodies / chemistry
  • Inclusion Bodies / metabolism
  • Recombinant Fusion Proteins / chemistry*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Solubility

Substances

  • Recombinant Fusion Proteins