Bioengineering of air-filled protein nanoparticles by genetic and chemical functionalization

Abstract

Background Various bacteria and archaea, including halophilic archaeon Halobacterium sp. NRC-1 produce gas vesicle nanoparticles (GVNPs), a unique class of stable, air-filled intracellular proteinaceous nanostructures. GVNPs are an attractive tool for biotechnological applications due to their readily production, purification, and unique physical properties. GVNPs are spindle- or cylinder-shaped, typically with a length of 100 nm to 1.5 μm and a width of 30–250 nm. Multiple monomeric subunits of GvpA and GvpC proteins form the GVNP shell, and several additional proteins are required as minor structural or assembly proteins. The haloarchaeal genetic system has been successfully used to produce and bioengineer GVNPs by fusing several foreign proteins with GvpC and has shown various applications, such as biocatalysis, diagnostics, bioimaging, drug delivery, and vaccine development. Results We demonstrated that native GvpC can be removed in a low salt buffer during the GVNP purification, leaving the GvpA-based GVNP's shell intact and stable under physiological conditions. Here, we report a genetic engineering and chemical modification approach for functionalizing the major GVNP protein, GvpA. This novel approach is based on combinatorial cysteine mutagenesis within GvpA and genetic expansion of the N-terminal and C-terminal regions. Consequently, we generated GvpA single, double, and triple cysteine variant libraries and investigated the impact of mutations on the structure and physical shape of the GVNPs formed. We used a thiol–maleimide chemistry strategy to introduce the biotechnological relevant activity by maleimide-activated streptavidin–biotin and maleimide-activated SpyTag003-SpyCatcher003 mediated functionalization of GVNPs. Conclusion The merger of these genetic and chemical functionalization approaches significantly extends these novel protein nanomaterials' bioengineering and functionalization potential to assemble catalytically active proteins, biomaterials, and vaccines onto one nanoparticle in a modular fashion. Graphical Abstract

A systematic analysis of affinity tags in the haloarchaeal expression system, Haloferax volcanii for protein purification

“…H. volcanii is an obligate halophilic archaeon, originally isolated from the Dead Sea, which naturally thrives in extreme conditions ( Strillinger et al, 2016 ; Grötzinger et al, 2018 ; Akal et al, 2019 ; Sysoev et al, 2021 ). H. volcanii has been equipped with a suite of microbiological and molecular genetics techniques, including an efficient DNA transformation system, shuttle plasmids, and selectable markers, making it a versatile platform for genetic studies, proteomics, and biotechnology research ( Allers, 2010 ; Strillinger et al, 2016 ; Haque et al, 2019 ; Pérez-Arnaiz et al, 2020 ; Karan et al, 2023 ). Moreover, H. volcanii grows, compared to other extreme halophiles, under laboratory culture conditions at high density and in a wide range of temperatures (37–55°C) and salt concentrations (1.8–3.5 M NaCl) ( Haque et al, 2019 , 2020 ; Sysoev et al, 2021 ).…”

“…Due to the fragile nature of the outer S-layer cell walls, H. volcanii cells easily lyse in hypotonic conditions, such as low salt buffer or water, releasing all cellular proteins ( Dyall-Smith, 2008 ). This allows a simplified downstream protein purification with minimal labor and cost, making H. volcanii attractive for biotechnology use ( Grötzinger et al, 2018 ; Akal et al, 2019 ; Karan et al, 2020 , 2023 ; Alshehri et al, 2022 ).…”

“…These changes serve a dual purpose: they reduce potential contaminants and enable polyhistidine-tag (His-tag) purification, a common method in protein purification. Moreover, H1895 has proven to be an excellent platform for expressing various enzymes and larger molecular structures ( Allers, 2010 ; Strillinger et al, 2016 ; Haque et al, 2019 , 2020 ; Karan et al, 2023 ). Furthermore, the lack of biofilm formation extends its utility to bioreactor applications, further underlining its versatility and potential for biotechnological endeavors ( Strillinger et al, 2016 ; Haque et al, 2020 ).…”