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

Cited 7 time in webofscience Cited 0 time in scopus
  • Hit : 418
  • Download : 0
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 A degrees 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.
Publisher
SPRINGER
Issue Date
2012
Language
English
Article Type
Article; Proceedings Paper
Keywords

RECOMBINANT HUMAN INSULIN; BACTERIAL INCLUSION-BODIES; FACTOR-I; CYCLODEXTRIN GLYCOSYLTRANSFERASE; PROTEIN; FUSION; PURIFICATION; BINDING; GENE

Citation

BIOPROCESS AND BIOSYSTEMS ENGINEERING, v.35, no.1-2, pp.255 - 263

ISSN
1615-7591
DOI
10.1007/s00449-011-0619-7
URI
http://hdl.handle.net/10203/94135
Appears in Collection
CBE-Journal Papers(저널논문)
Files in This Item
There are no files associated with this item.
This item is cited by other documents in WoS
⊙ Detail Information in WoSⓡ Click to see webofscience_button
⊙ Cited 7 items in WoS Click to see citing articles in records_button

qr_code

  • mendeley

    citeulike


rss_1.0 rss_2.0 atom_1.0