High-level production of a kringle domain variant by high-cell-density cultivation of <Emphasis Type="Italic">Escherichia coli</Emphasis> |
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Authors: | Seung Hoon Jang Chang Han Lee Yong Sung Kim Ki Jun Jeong |
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Institution: | (1) Department of Chemical and Biomolecular Engineering, KAIST, 335 Gwahagno, Yuseong-gu, Daejeon, 305-701, Republic of Korea;(2) Department of Molecular Science and Technology, Ajou University, San 5, Woncheon-dong, Yeongtong-gu, Suwon, 443-749, Republic of Korea;(3) Institute for the BioCentury, KAIST, 335 Gwahagno, Yuseong-gu, Daejeon, 305-701, Republic of Korea; |
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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). |
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