Biotechnology Letters

, Volume 31, Issue 5, pp 659–664

Single step intein-mediated purification of hGMCSF expressed in salt-inducible E. coli

  • K. Srinivasa Babu
  • T. Muthukumaran
  • Aju Antony
  • S. D. Prem Singh Samuel
  • M. Balamurali
  • V. Murugan
  • S. Meenakshisundaram
Original Research Paper

Abstract

Human granulocyte-macrophage colony stimulating factor (hGMCSF) is an important therapeutic cytokine. As a novel attempt to purify hGMCSF protein, without the enzymatic cleavage of the affinity tag, an intein-based system was used. The gene was fused by overlap extension PCR to the intein sequence at its N-terminal in pTYB11 vector. The hGMCSF was expressed as a fusion protein in E. coli BL21(DE3), and E. coli GJ1158. In the former, the protein was expressed as inclusion bodies and upon purification the yield was 7 mg/l with a specific activity of 0.5 × 107 IU/mg. In salt-inducible E. coli GJ1158, hGMCSF was expressed in a soluble form at 20 mg/l and a specific activity of 0.9 × 107 IU/mg. The intein-hGMCSF was purified on a chitin affinity column by cleaving intein with 50 mM DTT resulting in a highly pure 14.7 kDa hGMCSF.

Keywords

Escherichia coli GJ1158 Fusion protein expression Granulocyte macrophage colony stimulating factor Intein 

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Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • K. Srinivasa Babu
    • 1
  • T. Muthukumaran
    • 1
  • Aju Antony
    • 1
  • S. D. Prem Singh Samuel
    • 1
  • M. Balamurali
    • 1
  • V. Murugan
    • 1
  • S. Meenakshisundaram
    • 1
  1. 1.Centre for BiotechnologyAnna UniversityChennaiIndia

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