site > International journal > (2012) Effects of L-arginine on refolding of lysine-tagged human insulin-like growth factor 1 expressed in Escherichia coli.

Dept. of Advanced Fermentation Fusion Science & Technology, Kookmin University

|
회원가입

Topics

Professor | Current members | Alumni

Topics

International | Domestic | Patent

HOME > PUBLICATIONS

PUBLICATIONS

작성일 : 12-07-18 08:58

(2012) Effects of L-arginine on refolding of lysine-tagged human insulin-like growth factor 1 expressed in Escherichia coli.

글쓴이 : 박용철

추천 : 0

http://www.springerlink.com/content/a5212q286127t048/ [455]

Effects of L-arginine on refolding of lysine-tagged human insulin-like growth factor 1 expressed in Escherichia coli.

Seung Phill Choi, Yong-Cheol park, JungHwa Lee, Sang Jun Sim, and Ho-Nam Chang
Bioprocess and Biosystems Engineering. 35(1-2): 255-263. 2012.01.06
(SCI I/F: 1.809)

Insulin-like growth factor 1 (IGF1), a therapeutic protein, is highly homologous to proinsulin in 3-dimensional structure. To highly express IGF1 in recombinant Escherichia coli, IGF1 was engineered to be fused with the 6-lysine tag and ubiquitin at its N-terminus (K6Ub-IGF1). Fed-batch fermentation of E. coli TG1/pAPT-K6Ub-IGF1 resulted in 60.8 g/L of dry cell mass, 18% of which was inclusion bodies composed of K6Ub-IGF1. Subsequent refolding processes were conducted using accumulated inclusion bodies. An environment of 50 mM bicine buffer (pH 8.5), 125 mM l-arginine, and 4 °C was chosen to optimize the refolding of K6Ub-IGF1, and 240 mg/L of denatured K6Ub-IGF1 was refolded with a 32% yield. The positive effect of l-arginine on K6Ub-IGF1 refolding might be ascribed to preventing unfolded K6Ub-IGF1 from undergoing self-aggregation and thus increasing its solubility. The simple dilution refolding, followed by cleavage of the fusion protein by site-specific UBP1 and chromatographic purification of IGF1, led production of authentic IGF1 with 97% purity and an 8.5% purification yield, starting from 500 mg of inclusion bodies composed of K6Ub-IGF1, as verified by various analytical tools, such as RP-HPLC, CD spectroscopy, MALDI-TOF mass spectrometry, and Western blotting. Thus, it was confirmed that l-arginine with an aggregation-protecting ability could be applied to the development of refolding processes for other inclusion body-derived proteins.

Contact Us

Science Building Room No. 204-205, Kookmin University
861-1 Jeongrung-dong, Sungbuk-gu, Seoul 136-702
Tel: +82-2-910-5596(5462), Fax: +82-2-910-5739
E-mail: ycpark@kookmin.ac.kr