Engineering bacteria for the biosynthesis of complex protein complexes
Protein cages found naturally within microbes help their contents resist the harsh intracellular environment – an observation that applies to many bioengineering applications. Tokyo Tech researchers recently developed an innovative bioengineering approach using genetically modified bacteria; These bacteria can incorporate protein cages around protein crystals. This intracellular biosynthesis method efficiently produces highly customized protein complexes, which could find applications as advanced solid catalysts and functional nanomaterials.
In nature, proteins can assemble to form organized complexes with countless shapes and purposes. Thanks to remarkable advances in bioengineering over the past few decades, scientists can now produce customized protein combinations for specialized applications. For example, protein cages can trap enzymes that act as catalysts for a targeted chemical reaction, protecting it from a potentially harsh cell environment. Likewise, protein crystals—structures made up of repeating units of proteins—can serve as scaffolds for the fabrication of solids with exposed functional ends.
However, incorporating (or “encapsulating”) foreign proteins onto the surface of a protein crystal is challenging. Thus, the fabrication of protein crystals that encapsulate foreign protein aggregates has been elusive. To date, there are no effective methods to achieve this goal, and the types of protein crystals produced are limited. But what if bacterial cellular machinery could achieve this goal?
In a recent study, a research team from the Tokyo Institute of Technology, including Professor Takafumi Ueno, reported something new In the cell A method for encapsulating protein cages with various functionalities onto protein crystals. Their paper published in Nano messagesIt represents a major breakthrough in protein crystal engineering.
The team’s innovative strategy involves genetic modification Escherichia coli Bacteria produce two basic building blocks: polyhedral monomer (PhM) and modified ferritin (Fr). On the one hand, PhMs naturally combine within cells to form a well-studied protein crystal called polyhedral crystals (PhC). On the other hand, 24 Fr residues are known to combine to form a stable protein cage. “Ferritin has been widely used as a template for constructing bio-nanomaterials by modifying their inner and outer surfaces. Thus, if the formation of the Fr cage and its subsequent immobilization on PhC can be performed simultaneously in a single cell, the applications will be expanded,” explains Professor Ueno. Intracellular protein crystals as biohybrid materials.
To stabilize Fr cages in PhC, researchers modified the gene coding for Fr to include the α-helix(H1) tag of PhM, thus creating H1-Fr. The reason for this approach is that the naturally occurring H1 helices in PhM molecules interact significantly with tags on H1-Fr, acting as “recruitment factors” that bind foreign proteins to the crystal.
Using advanced microscopic, analytical and chemical techniques, the research team validated the proposed approach. Through various experiments, they found that the resulting crystals have a core-shell structure, i.e. a cubic core of PhC about 400 nanometers wide covered by five or six layers of H1-Fr cages.
This strategy for the biosynthesis of functional protein crystals holds much promise for applications in medicine, catalysis, and biomaterials engineering. “H1-Fr cages have the ability to immobilize external molecules inside them to deliver molecules,” says Professor Ueno. “Our results indicate that H1-Fr/PhC core-shell structures display H1-Fr cages on the outside of the cages.” “The PhC core can be individually controlled at the nanoscale. By assembling different functional molecules into the PhC core and the H1-Fr cage, hierarchical crystals controlled at the nanoscale can be constructed for advanced biotechnology applications.”
Future work in this area will help us realize the true potential of protein crystals and assemblies in bioengineering. With any luck, these efforts will pave the way to a healthier, more sustainable future.