Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 255-263 |
| Number of pages | 9 |
| Journal | Bioprocess and Biosystems Engineering |
| Volume | 35 |
| Issue number | 1-2 |
| DOIs | |
| Publication status | Published - 2012 Jan 1 |
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Keywords
- Inclusion body
- Insulin-like growth factor 1
- L-arginine
- Refolding
ASJC Scopus subject areas
- Biotechnology
- Bioengineering
Cite this
Effects of L-arginine on refolding of lysine-tagged human insulin-like growth factor 1 expressed in Escherichia coli. / Choi, Seung Phill; Park, Yong Cheol; Lee, Jung Hwa; Sim, Sang Jun; Chang, Ho Nam.
In: Bioprocess and Biosystems Engineering, Vol. 35, No. 1-2, 01.01.2012, p. 255-263.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Effects of L-arginine on refolding of lysine-tagged human insulin-like growth factor 1 expressed in Escherichia coli
AU - Choi, Seung Phill
AU - Park, Yong Cheol
AU - Lee, Jung Hwa
AU - Sim, Sang Jun
AU - Chang, Ho Nam
PY - 2012/1/1
Y1 - 2012/1/1
N2 - 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.
AB - 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.
KW - Inclusion body
KW - Insulin-like growth factor 1
KW - L-arginine
KW - Refolding
UR - http://www.scopus.com/inward/record.url?scp=84857448424&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84857448424&partnerID=8YFLogxK
U2 - 10.1007/s00449-011-0619-7
DO - 10.1007/s00449-011-0619-7
M3 - Article
VL - 35
SP - 255
EP - 263
JO - Bioprocess and Biosystems Engineering
JF - Bioprocess and Biosystems Engineering
SN - 1615-7591
IS - 1-2
ER -