This summer I took a course at Rutgers University in New Jersey called GFP: the Art and the Science. It was taught by a biochemistry professor, Dr. William Ward (who had started his own company, Brighter Ideas Inc., to help others realize the potential of GFP), and supposed to be quite difficult. I gleaned this by the topics we were required to write about to get into the program. A lot of them weren’t things that normal students would know off the top of their heads, and questions often had terms like ‘chromophore’ and ‘cyclic tripeptide’ in them. We were supposed to be investigating GFP, a relatively new protein.
Our task was to use biochemistry purification techniques to purify a crude sample of GFP. Dr. Ward and his assistants had inserted the gene for GFP production into e. coli, produced numerous bacteria colonies and put the colonies in to a blender. The result was a green, cloudy, viscous mixture of dead, disembodied bacteria, DNA, other proteins, and ribosomes, etc. These were referred to as ‘contaminants’. Because we only wanted the GFP this meant that we would have to get rid of everything else, which would be difficult. One of the techniques used was chromatography, which is a method for separating substances based on certain criteria, such as charge, polarity, hydrophobicity, density, size, shape, affinity, and solubility. We used ammonium sulfate precipitation, hydrophobic interaction chromatography (HIC), ion exchange chromatography (IEX), and high pressure liquid chromatography (HPLC) to purify the crude GFP sample. After each round of purification we used a spectrophotometer to measure the purity of our sample. We ended up purifying the crude sample, which was only about 25% GFP, to a sample that was nearly 100% GFP. But how to these purification techniques that we used work? Why are they so effective?
Nearly pure GFP on a UV light
samples of the purest GFP
collecting samples of GFP as it elutes from the chromatography column
Ammonium sulfate precipitation is a method used to purify GFP by altering its solubility. It solidifies GFP into a mass that does not easily dissolve in water. Therefore, when the crude GFP sample is centrifuged, the GFP separates from the more soluble contaminants by forming a pellet on the bottom. HIC purifies the sample by resulting in only the molecules that have a similar hydrophobicity. IEX purifies the sample by resulting in only molecules with a similar charge. Hydrophobicity and charge are two important characteristics of GFP. By purifying the crude GFP sample using by using these two criteria, a high level of success was ensured.
However, you may be asking yourself why GFP is important. If scientists are interested in purifying it, this must mean that it is worth isolating for further study or for further applications in real life. GFP is special simply because it glows green when exposed blue light. It first came to the public knowledge when a Japanese organic chemist and marine biologist, Osamu Shimamura, decided to investigate what allowed aequorea victoria jellyfish to glow green. He was the first person to isolate GFP from the jellyfish and find out which “part of GFP was responsible for its fluorescence” (Zimmer, Marc). He, along with Roger Tsien and Martin Chalfie won the Nobel Prize in Chemistry in the year 2008 for the “discovery and development of the green fluorescent protein GFP” (nobelprize.org, 2008), which only demonstrates the level of interest it has created in the scientific community. GFP can be modified for a large number of uses – it has been used to create glowing animals/pets, used as a marker in a cancer cell to track the activity of cancer in certain situations, and used in brains to create fluorescent multicolored neutrons which result in beautiful rainbow pictures of brain activity. It is even possible for GFP to be manipulated to express different colors. However, in order for GFP to be used in this fashion GFP must first be isolated, purified and studied, which is why the success of our purification was so important. Only by purifying it can the true potential of GFP be realized.
- “Martin Chalfie – Autobiography”. Nobelprize.org. 24 Jul 2011 http://nobelprize.org/nobel_prizes/chemistry/laureates/2008/chalfie.html
- Zimmer, Marc. “Green Fluorescent Protein – GFP History – Osamu Shimomura.” Connecticut College: Home Page. Web. 24 July 2011. <http://www.conncoll.edu/ccacad/zimmer/GFP-ww/shimomura.html>.
- Zimmer, Marc. “Green Fluorescent Protein – Cool Uses.” Connecticut College: Home Page. Web. 24 July 2011. < http://www.conncoll.edu/ccacad/zimmer/GFP-ww/cooluses0.html>.
The rainbow of GFP colors
glofish – genetically modified pet fish