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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

Paper ID Volume ID Publish Year Pages File Format Full-Text
17912 42705 2009 9 PDF Available
Title
Efficient proteolytic cleavage by insertion of oligopeptide linkers and its application to production of recombinant human interleukin-6 in Escherichia 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.

Keywords
Interleukin-6; Enzymatic cleavage; Enterokinase; Factor Xa; Thrombin; Maltose-binding protein
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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
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 44, Issue 5, 6 May 2009, Pages 254–262
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us