Efficient proteolytic cleavage by insertion of oligopeptide linkers and its application to production of recombinant human interleukin-6 in Escherichia coli
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- Efficient proteolytic cleavage by insertion of oligopeptide linkers and its application to production of recombinant human interleukin-6 in Escherichia coli
- Eun Gyo Lee; J E Baek; Seung-hui Lee; T W Kim; Jeong Ho Choi; Mun Chual Rho; Jungoh Ahn; Hong-Weon Lee; Joon Ki Jung
- Bibliographic Citation
- Enzyme and Microbial Technology, vol. 44, no. 6, pp. 254-262
- Publication Year
- 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.
- EnterokinaseEnzymatic cleavageFactor XaInterleukin-6Maltose-binding proteinThrombin
- Appears in Collections:
- Division of Bio Technology Innovation > BioProcess Engineering Center > 1. Journal Articles
Jeonbuk Branch Institute > Functional Biomaterial Research Center > 1. Journal Articles
Division of Bio Technology Innovation > 1. Journal Articles
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