Life’s a Great Balancing Act

By Jen Hu

“So be sure when you step.
Step with care and great tact
and remember that Life’s
a Great Balancing Act.
Just never forget to be dexterous and deft.
And never mix up your right foot with your left.
-Dr. Seuss’ “Oh the places you’ll go!”

Over the last few weeks of purification, remembering that “…Life’s a Great Balancing Act,” may be the only thing preserving my sanity. My project this summer is to crystallize a complex of the FTO (fat mass and obesity-associated) protein with a single strand oligomer of DNA. However, even before the crystallization process starts, one must isolate the protein. This is easier said than done.

From start to finish, each minute counts in purifying your protein. However, freshness and purity often work at odds with each other. One could have a fresher protein that has undergone less purification or an older protein that is extremely pure. There are three types of purifications that I have been working with: Nickel Affinity, Mono-Q, Gel Filtration.

Quick description of each:

To use the Nickel affinity column, your protein must have a histidine tag that was incorporated onto it during its insertion into the plasmid. The supernatant from the lysed bacteria is passed through the column and any protein with a His-tag sticks to the sides. This column provides the greatest specificity and is the fastest to run. It is my favorite column for a reason.

The Mono-Q column separates based on charge. Each protein has a pI, or isoelectric point, which represents the pH at which it has a neutral charge. Therefore, by using the buffer of an appropriate pH above or below the pI, one can separate proteins based on charge. Unfortunately, our column is somewhat broken and runs very slowly. On top of that, ordering scientific equipment in China takes longer than it should. Yikes.

The Gel Filtration column separates based on size. The larger the protein, the faster it flows through the column because there are fewer routes it can take through the gel. This is the most satisfying column because you can watch the UV absorbance reading and see your protein eluting out. I literally hold my breath and watch for the peak to emerge. It’s a bit funny the things that we live for in the world of science: a band on a gel, a peak on a graph, or a p-value less than 0.05. The UV peak(s) that I live for:

The first peak is likely a dimer of the protein of interest and therefore would elute out more quickly than the monomer, represented by the second peak.

As for optimizing the purification process, the main issue I’ve been debating is whether to deal with the problematic Mono-Q column or just let it go. In the end, I decided to let it go and replace the normal protocol of Nickel, Mono-Q, Gel Filtration with Nickel, Gel Filtration, Nickel. This optimization has given me the best purity and allows me to run the quick and specific Nickel column twice.

Optimization is no easy task. When you think about it, our entire lives are based on optimizations of happiness, health, and other factors. I’ll be honest: sometimes, the protein purification process becomes a question of whether I want to stay in the lab until 10pm or explore the city of Beijing. In Chicago, I don’t think I would mind staying late since I have all year to explore the city. However, opportunities such as working in a foreign country are hard to come by. As well, it doesn’t help that even a pure and fresh protein does not guarantee a good crystal: there are hundreds of screening conditions to do afterwards. The path of science is a long and arduous one, because if you think about it: there is no end. There is always more that can be done. However, I think we should strive to keep our lives balanced and keep working towards those optimizations, not relegate ourselves to accepting a life shut indoors without sunlight or proper nutrition. Optimizing the purification has not only given me a better protein, but also a better quality of life. For me, a better quality of life includes trying all the delicious food Beijing has to offer, such as jellyfish appetizers!