Terminal Amphipathic Peptide Induced In Vivo Enzyme Aggregates

Wednesday, November 10, 2010
Hall 1 (Salt Palace Convention Center)
Wei Wu, Lei Xing, Bihong Zhou, Zhen Cai, Bo Chen and Zhanglin Lin, Department of Chemical Engineering, Tsinghua University, Beijing, China

In recent years, a striking observation in the area of protein expression has been the discovery of biologically active inclusion bodies (AIBs) or protein aggregates1, which were largely believed to be inactive in the past. Several fusion partners including the foot-and-mouth disease virus VP1 capsid protein have been found to induce the formation of inclusion bodies of active enzymes2-4.

More strikingly, we recently found that even a short amphipathic peptide (18A) was able to promote efficient in vivo assembly of active protein aggregates in E. coli for several diverse model proteins, including Bacillus subtilis lipase A (LipA), Bacillus pumilus b-xylosidase (XynB), Aspergillus fumigatus amadoriase II (AMA), and green fluorescent protein (GFP). Except for GFP which was not quantitated, a majority of the total activities accumulated in the insoluble fractions (58%-97%), with the specific activities approaching those of the respective soluble native counterparts (42%-123%).

Laser scanning confocal micrograph (LSCM) and transmission electron micrograph (TEM) analyses of the cells expressing these aggregates showed that the AIBs  were deposited around the inside of the cells. Fourier transform infrared (FTIR) analyses of the aggregates revealed that the association was likely induced by intermolecular helical structures, not intermolecular cross-b structures, which was responsible for the formation of the AIBs reported earlier5.

Our work represents the first report where an amphipathic octadecapeptide can act as an inducer for in vivo assembly of active recombinant protein aggregates, which has potential applications in biocatalysis6-7 and high throughput purification and screening of intracellular proteins, and might be relevant to cellular protein complex assembly, and aggregation-related diseases8-9.

Reference

1       Gonzalez-Montalban, N., Garcia-Fruitos, E. & Villaverde, A. Recombinant protein solubility - does more mean better? Nat. Biotechnol. 25, 718-720, (2007).

2       Garcia-Fruitos, E. et al. Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins. Microb. Cell. Fact. 4, 6, (2005).

3       Arie, J.P., Miot, M., Sassoon, N. & Betton, J.M. Formation of active inclusion bodies in the periplasm of Escherichia coli. Mol. Microbiol. 62, 427-437, (2006).

4       Nahalka, J. & Nidetzky, B. Fusion to a pull-down domain: A novel approach of producing Trigonopsis variabilis D-amino acid oxidase as insoluble enzyme aggregates. Biotechnol. Bioeng. 97, 454-461, (2007).

5       De Groot, N.S., Sabate, R. & Ventura, S. Amyloids in bacterial inclusion bodies. Trends Biochem.Sci. 34, 408-416, (2009).

6       Burton, S.G., Cowan, D.A. & Woodley, J.M. The search for the ideal biocatalyst. Nat. Biotechnol. 20, 37-45, (2002).

7       Aehle, W. Enzymes in industry. (Wiley-VCH, Weinheim, Germany, 2004).

8       Kopito, R.R. Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol. 10, 524-530, (2000).

9       Bucciantini, M. et al. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 416, 507-511, (2002). 


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See more of this Session: Poster Session: Bioengineering
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division