Coiled-coil inspired functional inclusion bodies

Abstract

Background Recombinant protein expression in bacteria often leads to the formation of intracellular insoluble protein deposits, a major bottleneck for the production of soluble and active products. However, in recent years, these bacterial protein aggregates, commonly known as inclusion bodies (IBs), have been shown to be a source of stable and active protein for biotechnological and biomedical applications. The formation of these functional IBs is usually facilitated by the fusion of aggregation-prone peptides or proteins to the protein of interest, leading to the formation of amyloid-like nanostructures, where the functional protein is embedded. Results In order to offer an alternative to the classical amyloid-like IBs, here we develop functional IBs exploiting the coiled-coil fold. An in silico analysis of coiled-coil and aggregation propensities, net charge, and hydropathicity of different potential tags identified the natural homo-dimeric and anti-parallel coiled-coil ZapB bacterial protein as an optimal candidate to form assemblies in which the native state of the fused protein is preserved. The protein itself forms supramolecular fibrillar networks exhibiting only α-helix secondary structure. This non-amyloid self-assembly propensity allows generating innocuous IBs in which the recombinant protein of interest remains folded and functional, as demonstrated using two different fluorescent proteins. Conclusions Here, we present a proof of concept for the use of a natural coiled-coil domain as a versatile tool for the production of functional IBs in bacteria. This α-helix-based strategy excludes any potential toxicity drawback that might arise from the amyloid nature of β-sheet-based IBs and renders highly active and homogeneous submicrometric particles.

Coiled-Coil Based Inclusion Bodies and Their Potential Applications

“…To provide a suitable coiled-coil tag that preserves its native structure when embedded in IBs, our group performed a computational analysis of the biophysical properties of different IB-tags and selected the ZapB protein, a non-essential factor involved in the cell division process of E. coli (Ebersbach et al, 2008), as an optimal candidate for obtaining coiled-coil inspired IBs. ZapB is a homodimeric and anti-parallel coiled-coil domain able to self-assemble in vivo, and therefore, it allows the production of α-helix-rich IBs when fused to a target protein, as demonstrated by circular dichroism and Fourier transformed infrared spectroscopy (Gil-Garcia et al, 2020). ZapB was fused to two different fluorescent proteins in our study, obtaining active and biocompatible nanoparticles displaying native-like fluorescent spectra and devoid of any amyloid character.…”

The pre-induction temperature affects recombinant HuGM-CSF aggregation in thermoinducible Escherichia coli

“…The components of the secondary structure of IBs were identified by attenuated total reflection (ATR)–Fourier transform infrared (FTIR) spectroscopy (Valdez-Cruz et al 2017 ; Calcines-Cruz et al 2018 ; Singh et al 2020 ; Gil-Garcia et al 2020 ). Freshly purified IBs were dried at room temperature for 1 h using a speed vacuum concentrator and placed on Specac Quest ATR diamond accessory (Specac Limited, Slough, UK) coupled to an infrared spectrometer IRAffinity-1S (Shimadzu, Kyoto, Japan).…”

“…The amyloidogenic properties of IBs were evaluated by measuring the fluorescence after binding to the thioflavin T (Th-T) dye (Castellanos-Mendoza et al 2014 ; Calcines-Cruz et al 2018 ; Singh et al 2020 ; Gil-Garcia et al 2020 ). 50 mg/ml of protein in IBs were resuspended in phosphate buffer (pH 7.5) containing 75 µM of Th-T (Sigma Aldrich, St. Louis, MO, USA) and incubated for 1 h at 25 °C.…”

Methodological advances and strategies for high resolution structure determination of cellular protein aggregates

“…Many cell-derived aggregates, including bacterial IBs, show increased β character ( 68 , 69 ), and in some cases, amyloid-like features have been identified in cell-derived IBs using FTIR ( 68 , 70 ). More recently the coexistence of fibril-like intermolecular β-sheets with significant native structure ( 19 , 33 , 71 , 72 ) has further elucidated the heterogeneity of IBs. As the amount of residual native structure in IBs often correlates with the yield of enzymatically active protein, FTIR can be an essential screening tool for researchers trying to understand and optimize functional IBs ( 72 , 73 , 74 ).…”