(567r) Terminal Amphipathic Peptide Induced In Vivo Enzyme Aggregates

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 enzymes^2-4.

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 biocatalysis^6-7 and high throughput
purification and screening of intracellular proteins, and might be relevant to
cellular protein complex assembly, and aggregation-related diseases^8-9.

Reference

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2      
Garcia-Fruitos, E. et al. Aggregation as bacterial inclusion bodies does
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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).