Large-scale preparation of biologically active recombinant chicken obese protein (leptin)

Nina Raver, Mohammed Taouis, Sami Dridi, Michel Derouet, Jean Simon, Boaz Robinzon, Jean Djiane, Arieh Gertler*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

46 Scopus citations

Abstract

Prokaryotic expression vector pMON3401 encoding full size A(-1) chicken leptin (AF012727) was prepared by PCR of previously described cDNA. Escherichia coli cells transformed with this vector overexpressed large amounts of chicken leptin upon induction with nalidixic acid. The expressed protein found in the inclusion bodies was refolded and purified to homogeneity on a Q-Sepharose column, yielding two electrophoretically pure fractions (leptin-1 and leptin-2), eluted from the column by 100 and 125 mM NaCl. Both fractions showed a single band of the expected molecular mass of 16 kDa and were composed of over 95% of monomeric protein. The biological activity of both fractions, resulting from proper renaturation, was further evidenced by their ability to stimulate proliferation of leptin-sensitive BAF/3 cells transfected with a long form of human leptin-receptor construct and by lowering the food intake of starved chicken following intravenal or intraperitoneal injections.

Original languageEnglish
Pages (from-to)403-408
Number of pages6
JournalProtein Expression and Purification
Volume14
Issue number3
DOIs
StatePublished - Dec 1998

Keywords

  • Chicken
  • Leptin
  • Obese protein
  • Recombinant

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