Removal of endotoxin by reverse phase HPLC abolishes anti-endothelial cell activity of bacterially expressed plasminogen kringle 5

Biotechniques. 2003 Oct;35(4):724-6, 728, 730 passim. doi: 10.2144/03354st02.

Abstract

The success of recombinant protein expression/purification in Escherichia coli depends on a high-fidelity system rendering purified proteins free of confounding contaminants such as endotoxin. Here we report on the expression and purification of a cryptic plasminogen-derived domain, kringle 5, which was previously reported to specifically inhibit endothelial cell growth and, therefore, angiogenesis. Using a histidine (HIS)-tag expression and Ni(+)-NTA agarose purification system identical to previous reports, we found that our purified recombinant kringle 5 did inhibit endothelial cell growth, but this activity could not be eradicated by heat denaturing or proteolysis of kringle 5 with various proteases. This led us to suspect the presence of a contaminant in the purified samples. Quantitative endotoxin testing using a limulus amoebocyte lysate assay revealed that all samples purified by Ni(+)-NTA agarose alone harbored high concentrations of endotoxin that could not be removed by additional purification on anion exchange chromatography. Finally, when kringle 5 was rendered endotoxin-free by purification on reverse phase high-performance liquid chromatography (HPLC), there was a complete loss of endothelial cell growth inhibitory activity. These results strongly suggest that endotoxin-free recombinant kringle 5 may not possess anti-angiogenic activity and demonstrates that, especially in angiogenesis type assays, endotoxin contamination can lead to a misinterpretation of results.

Publication types

  • Comparative Study
  • Evaluation Study
  • Technical Report
  • Validation Study

MeSH terms

  • Animals
  • Artifacts
  • Cattle
  • Cell Survival / drug effects
  • Cells, Cultured
  • Chromatography, High Pressure Liquid / methods*
  • Drug Contamination / prevention & control*
  • Endothelial Cells / drug effects*
  • Endothelial Cells / physiology
  • Endotoxins / biosynthesis*
  • Endotoxins / isolation & purification*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Humans
  • Kringles
  • Plasminogen / biosynthesis*
  • Plasminogen / genetics
  • Plasminogen / isolation & purification
  • Plasminogen / pharmacology*
  • Quality Control
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / pharmacology

Substances

  • Endotoxins
  • Recombinant Proteins
  • endotoxin, Escherichia coli
  • Plasminogen