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Nucleic Acids Research 2005 33(12):3751-3762; doi:10.1093/nar/gki692
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Published online 8 July 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Article

Heterologous gene expression using self-assembled supra-molecules with high affinity for HSP70 chaperone

Ji-Young Ahn, Hyung Choi, Yang-Hoon Kim, Kyung-Yeon Han, Jin-Seung Park, Sung-Sik Han1 and Jeewon Lee*

Department of Chemical and Biological Engineering, Korea University Anam-Dong 5-1, Sungbuk-Ku, Seoul 136-713, South Korea 1School of Life Sciences and Biotechnology, Korea University Anam-Dong 5-1, Sungbuk-Ku, Seoul 136-713, South Korea

*To whom correspondence should be addressed. Tel: +82 2 3290 3304; Fax: +82 2 926 6102; Email: leejw{at}korea.ac.kr

Received March 24, 2005. Revised May 25, 2005. Accepted June 16, 2005.

Contrary to the results of direct expression, various human proteins (ferritin light-chain, epithermal growth factor, interleukin-2, prepro-ghrelin, deletion mutants of glutamate decarboxylase and arginine deiminase, and mini-proinsulin) were all soluble in Escherichia coli cytoplasm when expressed with the N-terminus fusion of ferritin heavy-chain (FTN-H). Through systematic investigations, we have found that a specific peptide motif within FTN-H has a high affinity to HSP70 chaperone DnaK, and that the peptide motif was composed of a hydrophobic core of three residues (Ile, Phe and Leu) and two flanking regions enriched with polar residues (Gly, Gln and Arg). It was also observed that all the recombinant proteins expressed with the fusion of FTN-H formed spherical nanoparticles with diameters of 10–15 nm, as confirmed by the transmission electron microscopy image. The protein nanoparticles are non-covalently cross-linked supra-molecules formed by the self-assembly function of FTN-H. Upon the formation of the supra-molecule, its size is likely to be limited by the assembly properties of FTN-H, thereby keeping the self-assembled particles soluble. This study reports on the dual function of FTN-H for fusion expression and solubility enhancement of heterologous proteins: (i) high-affinity interaction with DnaK and (ii) formation of self-assembled supra-molecules with limited and constant sizes, thereby avoiding the undesirable formation of insoluble macro-aggregates of heterologous proteins.


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