Coiled-Coil Based Inclusion Bodies and Their Potential Applications

Abstract

The production of recombinant proteins using microbial cell factories is frequently associated with the formation of inclusion bodies (IBs). These proteinaceous entities can be sometimes a reservoir of stable and active protein, might display good biocompatibility, and are produced efficiently and cost-effectively. Thus, these submicrometric particles are increasingly exploited as functional biomaterials for biotechnological and biomedical purposes. The fusion of aggregation-prone sequences to the target protein is a successful strategy to sequester soluble recombinant polypeptides into IBs. Traditionally, the use of these IB-tags results in the formation of amyloid-like scaffolds where the protein of interest is trapped. This amyloid conformation might compromise the protein’s activity and be potentially cytotoxic. One promising alternative to overcome these limitations exploits the coiled-coil fold, composed of two or more α-helices and widely used by nature to create supramolecular assemblies. In this review, we summarize the state-of-the-art of functional IBs technology, focusing on the coiled-coil-assembly strategy, describing its advantages and applications, delving into future developments and necessary improvements in the field.

Protein-only Nanoparticles for T Cell Expansion and Activation

“…ccNPs are reminiscent of bacterial inclusion bodies (IBs) but differ in their assembly mechanism. Unlike conventional IBs, ccNPs are not sustained by the formation of intermolecular β-sheets of an amyloid-like nature between misfolded and thus mostly inactive polypeptide chains, but instead by the assembly of native α-helical structures, which are not expected to significantly impact the fold of the adjacent proteins . Consistent with this view, purified ccNPs are highly fluorescent and display fluorescence excitation and emission spectra equivalent to those of native GFP (Figure f,g).…”

“…Unlike conventional IBs, ccNPs are not sustained by the formation of intermolecular βsheets of an amyloid-like nature between misfolded and thus mostly inactive polypeptide chains, 28 but instead by the assembly of native α-helical structures, which are not expected to significantly impact the fold of the adjacent proteins. 29 Consistent with this view, purified ccNPs are highly fluorescent and display fluorescence excitation and emission spectra equivalent to those of native GFP (Figure 2f,g). Additionally, the Fourier transform infrared (FTIR) spectroscopy spectrum in the in the amide I region (1600−1700 cm −1 ) show a dominant signal at 1648 cm −1 , which can be attributed to the α-helical structures of the ZapB and Z domains, and which is absent or minor in conventional IBs 30 (Figure 2h).…”

Carrier‐Free Enzyme Immobilizates for Flow Chemistry

“…Due to their heterogenous nature, the structures of IBs and CatIBs are difficult to access, but studies using different spectroscopic methods have shown that their internal structure also depends on the aggregation inducing tags and can possess or be devoid of amyloid-like structures [96], and can contain a variable degree of unfolded polypeptides alongside natively folded protein [97][98][99]. Like IBs, CatIBs can easily be obtained by simple heterologous production in, e.g., E.coli, cell lysis and subsequent centrifugation and washing steps, yielding relatively pure (up to 90 % target protein) preparations (Fig.…”

Construction of a Protein Crystalline Inclusion-Based Enzyme Immobilization System for Biosynthesis of PAPS from ATP and Sulfate

“…Enzyme immobilization confines the soluble enzymes on insoluble carriers through physical or chemical methods. , Considering that the catalytic performance of enzymes is also impacted by the properties of carrier materials and the roles of chemical reagents, the laborious screening for suitable carriers or chemical reagents is always indispensable. To facilitate the immobilization process, many molecular immobilization strategies have been proposed recently. − In particular, many auto-assembly tags such as 18A, ELK16, L6KD, GFIL8, NSPdoT, HVdoT, and DMdoT have been developed for triggering the self-assembly of enzymes in vivo . Recently, Wang et al reported CipA and CipB from Photorhabdus luminescens as universal scaffolds for organizing enzymes into protein crystalline inclusions (PCIs) by N-terminal fusion .…”