Scientists at Tokyo Tech have developed a new cell-free protein crystallization (CFPC) method that is expected to advance research in structural biology. The technique will enable the analysis of unstable proteins that could not be studied using conventional methods, say the scientists, who add that analyzing these types of proteins will increase our knowledge of cellular processes and functions.

The team published its study “Cell-free protein crystallization for nanocrystal structure determination” in Scientific Reports.

Microscopic protein crystals are found in living cells and help sustain processes like immune system activation, protein storage, and protection. To better understand the relationship between protein crystals’ structure and function, in-cell protein crystallization (ICPC) has been used to directly observe protein crystals in living cells to ensure high-quality crystals without the need for purification processes or complex screening methods.

Schematic illustration of the CFPC process using a wheat germ protein synthesis kit to synthesis polyhedrin monomer (PhM), which was further crystallized to nano-sized polyhedra crystals. [Takafumi Ueno, PhD/Tokyo Tech]
However, despite ICPC’s many advantages, few structures were reported because the crystals formed in living cells didn’t have the size and quality that was required for analysis, according to a research team from Japan led by Takafumi Ueno, PhD, of Tokyo Tech. They set their sights on developing a better method that would make protein crystallization and analysis more efficient and effective. The result—CFPC—is a hybrid between in vitro protein crystallization and ICPC and reportedly allows rapid and direct formation of protein crystals without the need for complicated crystallization and purification methods.

“ICPC is expected to become an important tool in crystal structure analysis but we need a method to obtain better resolution protein crystal structures. So, we focused on establishing high-quality protein crystallization using CFPC with small-scale and rapid reactions,” notes Ueno, who heads his own lab.

“We have succeeded in crystallization and structure determination of nano-sized polyhedra crystal (PhC) at a high resolution of 1.80 Å. Furthermore, nanocrystals were synthesized at a reaction scale of only 20 μL using the dialysis method, enabling structural analysis at a resolution of 1.95 Å. To further demonstrate the potential of CFPC, we attempted to determine the structure of crystalline inclusion protein A (CipA), whose structure had not yet been determined,” write the investigators.

“We added chemical reagents as a twinning inhibitor to the CFPC solution, which enabled us to determine the structure of CipA at 2.11 Å resolution. This technology greatly expands the high-throughput structure determination method of unstable, low-yield, fusion, and substrate-biding proteins that have been difficult to analyze with conventional methods.”

“The high-quality protein crystals produced by our method will expand the horizons of structural determination and provide us with useful and unprecedented insights into the complex environment of living cells,” said Ueno.