title: High-level production of a kringle domain variant by high-cell-density cultivation of Escherichia coli.
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 (approximately 5.4 g/L) were significantly enhanced to values that were much higher than those found previously with Pichia cultivation (<8 mg/L).
authors.affiliation: Department of Chemical and Biomolecular Engineering, KAIST, 335 Gwahagno, Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors.forename: Seung Hoon
authors.initials: SH
authors.lastname: Jang
authors.forename: Chang Han
authors.initials: CH
authors.lastname: Lee
authors.forename: Yong Sung
authors.initials: YS
authors.lastname: Kim
authors.forename: Ki Jun
authors.initials: KJ
authors.lastname: Jeong
mesh_terms: D001798:Blood Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D018082:Kringles; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D001798:Blood Proteins; D011994:Recombinant Proteins