Membrane Protein Expression
The challenge of overexpressing membrane proteins in cells needs to be addressed on multiple levels. First, can you make enough protein without killing the cells? Second, what detergent provides optimal extraction of the membrane protein and stabilizes the protein without aggregation? (Membrane proteins often display significant nonspecific aggregation.)
Third, concentration of the protein enables formation of the proper crystal lattice structure. At concentrations of 1 mg/mL the protein solution is so dilute that the probability of forming crystal nuclei (the initial event in crystallization) is extremely low. The majority of proteins crystallized have initial concentrations between 5 and 20 mg/mL.
At higher concentrations in homogeneous solutions the proteins have a higher probability of interacting in the proper orientation to form a proper crystal lattice (nucleation event). Unfortunately, for many aqueous and membrane proteins, as the concentration of purified protein is increased, there is often an increase in the formation of nonspecific aggregation producing a nonhomogeneous population of purified protein—not desirable for crystallization.
Self-interaction chromatography is a method that has been used to assess the best conditions to place a soluble protein that will support stability of the protein in high concentration without formation of aggregates. Development of an automated analytical self-interaction chromatography system is now being adopted for identification of buffer conditions that will support rapid identification of the optimum solubility conditions followed by identification of solution conditions with a higher probability of crystal formation.
“We’ve taken this same approach to determine the best buffer conditions to grow membrane protein crystals. We load the column with 0.75 mg of protein, allowing it to form covalent bonds to a polymer-based bead matrix in the column. This guarantees that every surface of the protein is exposed to the solvent,” said Larry DeLucas, Ph.D., director, Center for Structural Biology, University of Alabama at Birmingham.
“We then load a few µg of the same protein onto the column under 100 different buffer conditions that contain different solubilizing additives (excipients) and measure the elution retention time for the injected protein. This represents a small random sampling of the total number of possible combinations and concentrations that could be investigated.
“This experimental data along with other parameters, including total protein covalently bound to the matrix and the column void volume is input into an equation that calculates for each solution condition the second virial coefficient, a thermodynamic term that represents the sum of all protein-protein interactions. The calculated second virial coefficient values with their respective solution conditions are then input into an artificial neural network (ANN),” he continued.
“The ANN uses this input data to predict the second virial coefficient values for the complete factorial of possible solution conditions. The resulting predicted values provide an assessment of each solution condition’s ability to reduce (positive value) or increase (negative value) protein-protein interactions. For protein crystallization, we are looking for a gentle net attraction, solution parameters that force the protein molecules to display weak attraction, thereby improving the probability of forming high-quality crystals.”
From this approach the DeLucas lab has determined that lithium chloride in solutions up to 1 M can support the concentration of the cystic fibrosis transmembrane regulator protein from 0.05 mg/mL to 0.5 mg/mL in a homogeneous solution without formation of aggregates.
As a former payload specialist who flew aboard the 1992 U.S. Space Shuttle Columbia, mission STS-50, Dr. DeLucas was able to demonstrate that certain protein crystals are of higher quality when grown in a microgravity environment as compared with earth-grown crystals of the same protein. At that time, the challenge was the short duration of time in space.
Crystals grow slower in space and so the size of the crystals from the typical space shuttle 10-day mission was often too small for diffraction analysis. With the establishment of the International Space Station (ISS) the time constraints can be overcome. Dr. DeLucas will be contributing more than 100 proteins from researchers in academia and industry to a future commercial launch of the Space X Dragon laboratory, flown on the Falcon rocket.