Coiled-coil inspired functional inclusion bodies | JoVE Visualize

Area of Science:

Biotechnology
Protein Engineering
Biophysics

Background:

Recombinant protein expression in bacteria frequently results in insoluble inclusion bodies (IBs), hindering the production of active proteins.
Functional IBs, often amyloid-like, can be engineered for biotechnological applications by fusing proteins to aggregation-prone peptides.
These amyloid-like IBs embed the protein of interest within nanostructures.

Purpose of the Study:

To develop an alternative strategy for generating functional inclusion bodies (IBs) that avoids the use of amyloid structures.
To explore the potential of coiled-coil protein folds for creating stable, functional bacterial IBs.
To demonstrate the efficacy of a novel coiled-coil-based approach for producing active recombinant proteins.

Main Methods:

In silico analysis of protein tags for coiled-coil propensity, aggregation, charge, and hydropathicity.
Identification and utilization of the bacterial coiled-coil protein ZapB as a self-assembling tag.
Fusion of recombinant proteins to ZapB to form functional, non-amyloid IBs.
Characterization of IB structure (secondary structure analysis) and functional assessment of embedded proteins (using fluorescent proteins).

Main Results:

The bacterial coiled-coil protein ZapB was identified as an optimal candidate for forming functional IBs.
ZapB self-assembles into fibrillar networks with predominantly alpha-helix secondary structure, avoiding beta-sheet amyloid formation.
Functional IBs were successfully generated, preserving the native state and activity of fused recombinant proteins, as demonstrated with fluorescent proteins.
The resulting IBs were submicrometric, homogeneous, and non-toxic.

Conclusions:

A proof-of-concept for using a natural coiled-coil domain (ZapB) to produce functional inclusion bodies in bacteria was established.
This alpha-helix-based strategy offers a safer alternative to beta-sheet-based amyloid IBs, mitigating potential toxicity.
The method yields highly active and homogeneous submicrometric particles suitable for biotechnological applications.