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Title 

Efficient proteolytic cleavage by insertion of oligopeptide linkers and its application to production of recombinant human interleukin-6 in Escherichia coli

Authors 

Eun Gyo LeeJ E BaekSeung-hui LeeT W KimJeong Ho ChoiMun Chual RhoJungoh AhnHong-Weon LeeJoon Ki Jung

Publisher 

Elsevier

Issue Date 

2009

Citation 

Enzyme and Microbial Technology, vol. 44, no. 6, pp. 254-262

Keywords 

enterokinaseenzymatic cleavagefactor xainterleukin-6maltose-binding proteinthrombinescherichia coli

Abstract 

Efficient expression and purification of bioactive recombinant human interleukin-6 (hIL6) was successfully achieved in Escherichia coli (E. coli) by fusion of the maltose-binding protein (MBP) with hIL6 and the insertion of oligopeptide linkers. MBP/hIL6 was over-expressed in the soluble form at a concentration of approximately 2.5 g/L. For hIL6 recovery, enterokinase, factor Xa, and thrombin were employed to cleavage MBP from the fusion constructs. However, undesired and non-specific cleavage fragments as well as rhIL6 were obtained following the cleavage. The introduction of oligopeptide linkers at the C-terminal end of the fusion construct could improve the efficiency and the rate of the enzymatic cleavage reaction, and the rhIL6 purification was achieved by using MBP affinity chromatography, factor Xa cleavage, and reverse-phase chromatography, resulting in an overall yield as high as 33% (equivalent to 0.27 g hIL6/L) at purity over 98%. The biological activity of the purified recombinant hIL6 was demonstrated by confirming the presence of the signal transducer and activator of transcription 3 (STAT3) signaling pathway. This study suggests that the optimized peptide linker specifically designed for both fusion partner and target molecule has a great potential for efficient recombinant protein production.

ISSN 

0141-0229

Link 

http://dx.doi.org/10.1016/j.enzmictec.2008.12.014

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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