Expression, purification, and characterization of recombinant human interleukin 24 in Escherichia coli

Protein Expr Purif. 2007 Jun;53(2):339-45. doi: 10.1016/j.pep.2006.12.021. Epub 2007 Jan 4.

Abstract

Interleukin-24 (IL-24) can induce apoptosis of a broad range of tumor cells, and this function of IL-24 is independent of classic tumor suppressor genes, such as p53, Rb and p16. Here, we report the expression, purification and preparation of a recombinant IL-24 protein (rIL-24) without post-translational modifications, which may selectively induce apoptosis of tumor cells in vitro. We found that non-fusion rIL-24 was not able to be expressed by vectors pET11c, 28a, and 22b in Escherichia coli. To obtain recombinant non-fusion IL-24 protein, the encoding region for IL-24 was cloned between KpnI and BamHI in pET32a. The Trx (Thioredoxin)/IL-24 fusion proteins were expressed in the form of inclusion bodies in E. coli host strain BL21 (DE21). The expression level was more than 30% of total cell lysate. Inclusion bodies were disrupted, washed, and isolated at pH 9.0, and were completely dissolved in a buffer containing 2M urea at pH 9.0. After nickel ion metal affinity chromatography, gel filtration chromatography, and renaturation, the refolded fusion proteins with a purity of >96% were obtained. Trx/IL-24 proteins were digested by enterokinase (EK) to both Trx and rIL-24 fragments which then were separated by cation exchange chromatography. Cell proliferation experiments proved that the rIL-24 (98% purity) retains its cancer-selective apoptosis-inducing properties. This result suggested that the rIL-24 may have cancer therapeutic applications.

MeSH terms

  • Base Sequence
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • Cells, Cultured
  • DNA Primers / genetics
  • Escherichia coli / genetics*
  • Gene Expression
  • Humans
  • In Vitro Techniques
  • Interleukins / genetics*
  • Interleukins / isolation & purification*
  • Interleukins / pharmacology
  • Plasmids / genetics
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / pharmacology

Substances

  • DNA Primers
  • Interleukins
  • Recombinant Fusion Proteins
  • interleukin-24