earticle

영어

Human kringle domains (KDs) are ubiquitous domains known as binding modulator for various biomolecules with seven flexible loops, and very recently it was successfully demonstrated that KDs could be engineered toward target-specific binding proteins as an alternative protein scaffold. Here, we report the development of efficient expression system of KD derivative (KD548) as a promising anti-cancer agent in Escherichia coli host and fed-batch cultivation for its preparative scale production. Even though KD548 needs three disulfide bonds for correct folding, expression in cytoplasm allowed the highly soluble and much enhanced production than periplasm. For the efficient expression, four sets of expression systems consisting of different promoters (lac or T7) and different fusion tag (10xHis or FLAG) were examined. Among them, use of T7 promoter-FLAG tag fusion system resulted in much higher production than others in shake flask cultivation as well as in fed-batch cultivation performed in 6.6 L jar bioreactor. When cells were induced at higher cell density (OD600=100) and complex feeding solutions were supplemented, cells density and production yield could be improved significantly which were also much higher than that by previous Pichia host (~ 8 mg/L).