Refolding and purification process of therapeutic peptide IGF-1 by on-column and AOB methods = 컬럼상과 AOB 방법에 의한 의료용 펩타이드 IGF1의 재접힘과 정제공정

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Insulin-like growth factor 1 (IGF1) is a well-known therapeutic protein and highly homologous to proinsulin in three dimensional structure. A truncated IGF1 containing 70 amino acids from $Gly_{33}$ to $Ala_{102}$ was expressed in recombinant Escherichia coli. IGF1 was fused with the 6 lysine tag and ubiquitin at its N-terminal (K6Ub-IGF1) for soluble expression and easy purification. Fed-batch fermentation of E. coli TG1 containing the IGF1 expression system pAPT-K6Ub-IGF1 resulted in 60.8 g/L cell density and 18% K6Ub-IGF1 content relative to total protein. The fusion protein was mainly expressed as form of insoluble inclusion bodies with IPTG induction. High accumulation of K6Ub-IGF1 as inclusion body required efficient and economic refolding processes. The conditions of refolding were optimized through the dilution methods. At the optimal refolding condition of 50 mM bicine buffer (pH 8.5) containing 125 mM L-arginine and 0.25 mM L-cysteine, 240 mg/L of denatured K6Ub-IGF1 was refolded by 33% yield. Simple dilution refolding under the optimized condition, affinity chromatographic purification and site-specific UBP1 cleavage led to 97% of purity and 18.2% of total IGF1 purification yield based on the amount of inclusion body initially added. It was also found that the addition of L-arginine prevented self-aggregation of K6Ub-IGF1 and hence improved final refolding yield. The renaturation efficiency of the reduced IGF-I into the native conformation was greatly increased in the presence of 125mM L-arginine in 50mM bicine buffer, pH 8.5. The refolded fusion protein was selectively cleaved with UBP at the linkage site, without the degradation by peptidases derived from the host cell. The released IGF-I was purified using cation-exchange chromatography and reversed phase HPLC to give recombinant IGF-I of more than 97% purity. The optimized conditions of refolding and cleavage reactions were applid to the on-coumn process of cation exchange chromatography. ...
Advisors
Chang, Ho-Namresearcher장호남researcher
Description
한국과학기술원 : 생명화학공학과,
Publisher
한국과학기술원
Issue Date
2008
Identifier
303594/325007  / 000945445
Language
eng
Description

학위논문(박사) - 한국과학기술원 : 생명화학공학과, 2008. 8., [ ix, 118 p. ]

Keywords

IGF1; refolding; arginine; oleosin; 인성장호르몬; 재접힘; 아르기닌; 올레오신; IGF1; refolding; arginine; oleosin; 인성장호르몬; 재접힘; 아르기닌; 올레오신

URI
http://hdl.handle.net/10203/29048
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=303594&flag=dissertation
Appears in Collection
CBE-Theses_Ph.D.(박사논문)
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