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Applied Microbiology and Biotechnology

, Volume 92, Issue 2, pp 327–336 | Cite as

High-level production of a kringle domain variant by high-cell-density cultivation of Escherichia coli

  • Seung Hoon Jang
  • Chang Han Lee
  • Yong Sung Kim
  • Ki Jun JeongEmail author
Applied Genetics and Molecular Biotechnology

Abstract

Human kringle domains (KDs) are ubiquitously expressed binding modulators that fold into seven flexible loops and it has been previously demonstrated that KDs can be engineered toward target-specific binding proteins as a non-antibody protein scaffold. Here, we report a method for efficient expression of a KD derivative (KD548)—a promising anti-cancer agent—by high-cell-density culture of Escherichia coli at a preparative scale production. The correct folding of KD548 requires three disulfide bonds. Nevertheless, cytoplasmic expression of KD548 in E. coli led to good yields of highly soluble proteins with high activity. For efficient expression, four sets of expression systems consisting of different promoters (lac or T7) and fusion tags (His or FLAG) were examined. Of these, the expression system using a combination of the T7 promoter with the FLAG tag resulted in the highest production in shake flask cultivation as well as in high-cell-density cultivation performed in a 6.6-L jar bioreactor. When protein expression was induced at high-cell density (optical density [OD] = 100) and when complex feeding solutions were supplemented, cell density (maximum OD = 184) and production yield (∼5.4 g/L) were significantly enhanced to values that were much higher than those found previously with Pichia cultivation (<8 mg/L).

Keywords

Kringle domain Escherichia coli High-cell-density culture Fusion tag 

Notes

Acknowledgement

This research was supported by the Conversing Research Center Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant no. 2009-0082332).

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Seung Hoon Jang
    • 1
  • Chang Han Lee
    • 2
  • Yong Sung Kim
    • 2
  • Ki Jun Jeong
    • 1
    • 3
    Email author
  1. 1.Department of Chemical and Biomolecular EngineeringKAISTDaejeonRepublic of Korea
  2. 2.Department of Molecular Science and TechnologyAjou UniversitySuwonRepublic of Korea
  3. 3.Institute for the BioCenturyKAISTDaejeonRepublic of Korea

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