Electrostatic interaction-induced inclusion body formation of glucagon-like peptide-1 fused with ubiquitin and cationic tag

Protein Expression and Purification
Sung-Gun KimJin-Ho Seo

Abstract

In an attempt to produce glucagon-like peptide-1 (GLP-1) using recombinant Escherichia coli, ubiquitin (Ub) as a fusion partner was fused to GLP-1 with the 6-lysine tag (K6) for simple purification. Despite the high solubility of ubiquitin, the fusion protein K6UbGLP-1 was expressed mainly as insoluble inclusion bodies in E. coli. In order to elucidate this phenomenon, various N- and C-terminal truncates and GLP-1 mutants of K6UbGLP-1 were constructed and analyzed for their characteristics by various biochemical and biophysical methods. The experiment results obtained in this study clearly demonstrated that the insoluble aggregation of K6UbGLP-1 was attributed to the electrostatic interaction between the N-terminal 6-lysine tag and the C-terminal GLP-1 before the completion of folding which might be one of the reasons for protein misfolding frequently observed in many foreign proteins introduced with charged amino acid residues such as the His tag and the protease recognition sites. The application of a cation exchanger for neutralizing the positive charge of the 6-lysine tag in solid-phase refolding of K6UbGLP-1 successfully suppressed the electrostatic interaction-driven aggregation even at a high protein concentration, resul...Continue Reading

References

Apr 1, 1989·Proceedings of the National Academy of Sciences of the United States of America·T R ButtS T Crooke
Mar 1, 1994·Proteins·P L WintrodeP L Privalov
Jul 16, 1999·The EMBO Journal·S C JohnstonC P Hill
Sep 29, 1999·Proceedings of the National Academy of Sciences of the United States of America·M P Morrissey, E I Shakhnovich
Oct 11, 2002·Proceedings of the National Academy of Sciences of the United States of America·Fabrizio ChitiChristopher M Dobson
Jan 9, 2004·Protein Expression and Purification·Arun K Mohanty, Michael C Wiener
Oct 27, 2004·Journal of Structural and Functional Genomics·Esmeralda A WoestenenkHelena Berglund
Apr 29, 2005·Protein Engineering, Design & Selection : PEDS·Heather M Went, Sophie E Jackson
Mar 1, 2006·Trends in Biotechnology·Salvador Ventura, Antonio Villaverde
Jun 27, 2006·Protein Expression and Purification·Mareike KurzPawel Listwan
Mar 23, 2011·Protein Expression and Purification·Sung-Gun KimJin-Ho Seo

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Citations

May 1, 2013·Biological Chemistry·Hauke LilieFranziska Gehle
Jan 30, 2020·Applied Microbiology and Biotechnology·Mi-Ran Ki, Seung Pil Pack
Jun 9, 2018·Microorganisms·Vasiliki Paraskevopoulou, Franco H Falcone

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