Make It Glow
Many proteins of therapeutic interest are membrane bound. By definition these mostly hydrophobic molecules are normally surrounded by a lipid bilayer. Obtaining sufficient quantities of purified, properly folded protein from solubilized cellular membranes can present a great challenge.
When Genentech structural biology scientist Christopher Koth’s group is presented with a new protein target, the protein must be expressed to sufficient levels, purified, and then its high-resolution structure determined through crystallization or NMR.
The process usually involves generating a large number of constructs. This is because they typically want to determine the minimal active fragment of the protein suitable for structure studies or test mutations that may stabilize the purified protein. Often, the native full-length proteins simply won’t express. And they have many targets to evaluate.
“We run into this problem of having an early bottleneck, where we try to screen and identify those constructs that are most likely to yield us high-resolution crystal structures, or be amenable to structural studies by NMR.”
What if they could determine the suitability of an expressed protein for structural studies before purification? Perhaps it could be made visible in the context of all the other proteins in a cell—like looking for a bright yellow four leaf clover in a field of green clover—and there was a measure of how amenable it might be to those structural studies?
Fusing expressed proteins to a fluorescent protein, like GFP, is one possible solution. However, Genentech uses a standard HIS tag, and Koth didn’t want to change any of the cloning or expression pipeline. So, he and chemist Zachary Sweeney used a fluorophore that attaches to the HIS tag, as a result they were able to visualize the proteins as they came through a size exclusion column (SEC).
Experience has shown that proteins that give a good SEC chromatography profile may crystallize. “On the other hand, proteins that give a lousy profile almost never crystallize,” he explained. “Multiple peaks, asymmetric peaks, or peaks in the wrong spot—if we see that, we drop the construct and don’t proceed to purification.”
Koth uses a silica-based size-exclusion resin that can be run at very high flow rates. “We can do one run every approximately 20 minutes, so overnight we can screen a very large number of constructs,” he explained.
For some challenging targets, like membrane proteins, maybe only 10–20 constructs from a 96-well plate will behave well by the SEC test. While that may not seem like such a magic bullet assay—especially since there are no guarantees that they will indeed crystallize—it represents “a significant time savings for us,” Koth noted. It means “we can screen more constructs, and that increases our chances of coming up with something that’s going to give a high-resolution structure.”