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Title 

Enhanced protease cleavage efficiency on the glucagon-fused interleukin-2 by the addition of synthetic oligopeptides

Authors 

S W KimJ B KimWeon Sup LeeW H JungJ M RyuH W JangY B JoJoon Ki JungJ H Kim

Publisher 

Elsevier

Issue Date 

2007

Citation 

Protein Expression & Purification, vol. 55, no. 1, pp. 159-165

Keywords 

cleavage efficiencyenterokinasefactor Xafusion proteininterleukin-2glucagoninterleukin 2oligopeptides

Abstract 

Human interleukin-2 (hIL-2) was produced as a recombinant fusion protein (G3·IL-2/HF) consisting of three tandem-arranged human glucagon molecules (G3) and hIL-2. For the recovery of hIL-2, a factor Xa (FXa) cleavage sequence was introduced next to the N-terminus of hIL-2. Cleavage efficiency on this recombinant protein construct was very low because its recognition sequence was sterically hindered within the G3·IL-2/HF molecule and hence FXa access to the cleavage site was insufficient. We therefore introduced various synthetic oligopeptides upstream from the FXa cleavage site as a means to change substrate conformation and thereby increase cleavage efficiency. Among these oligopeptides, acidic or nucleophilic constructs were the most effective for the FXa-mediated cleavage of the fusion protein. In addition, insertion of various oligopeptides into the G3·IL-2/HF molecule varied the solubility of each construct depending on their physical properties. Consequently, the G3·IL-2/DF construct showed the highest final hIL-2 yields via FXa-mediated removal of the fusion partner. Lastly, we confirmed that cleavage efficiency was greatly increased but native hIL-2 was cleaved internally by non-specific cleavage when the acidic oligopeptide D4 (DDDD) was introduced upstream of the EK cleavage site within G3·IL-2/HE molecule. The G3·IL-2/HE molecule was shown to be an inefficient substrate to EK in a previous report (Biotechnol. Bioprocess Eng. (2000) 5, 13-16).

ISSN 

1046-5928

Link 

http://dx.doi.org/10.1016/j.pep.2007.04.003

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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