Professional Documents
Culture Documents
Patrick McKay
Senior Research Associate, Department of Recovery Sciences, Genentech, Inc.
A Personal Perspective
When people ask me about science, I try to explain to them that science is both fun and
interesting. That was what drew me to choose a career in a scientific field many years
ago. You don't want to be stuck in a job that is dull and boring for the rest of your life. As
a scientist employed in biotechnology, I've never had the feeling of being bored, or that
my scientific applications were dull. On the contrary, science to me is exciting and a
tremendous challenge. My job involves the purification of recombinant DNA-derived
proteins to a level of purity which will allow them to be used as therapeutic
biopharmaceuticals. The whole design of the purification process which is needed to
produce the protein is part of the challenge -- what step goes where, how do the steps
interface, etc. Troubleshooting these processes is similar to playing a detective like
Columbo -- you know that something is amiss, but what are the causes? Finally, being
able to scale-up this purification process to enable our Manufacturing group to carry out
these processes under GMP (Good Manufacturing Practices) conditions is a must if the
FDA (Food and Drug Administration) is to approve the product. There's nothing quite
like the satisfaction that a process chromatographer gets when his or her process is put
into place, and the recombinant protein is ultimately used to treat patients, some of whom
have life-threatening illnesses. Yes, science is fun and exciting. And, being a process
scientist in the biotechnology industry provides a researcher with challenges above and
beyond his or her wildest imaginations from what they learn in school. The following
paper describes some of the techniques that I use in my daily job, and may give you an
idea on how to integrate the field of chromatography into your biology classes.
What is Chromatography?
Chromatography is the science which is studies the separation of molecules based on
differences in their structure and/or composition. In general, chromatography involves
moving a preparation of the materials to be separated - the "test preparation" - over a
stationary support. The molecules in the test preparation will have different interactions
with the stationary support leading to separation of similar molecules. Test molecules
which display tighter interactions with the support will tend to move more slowly through
the support than those molecules with weaker interactions. In this way, different types of
molecules can be separated from each other as they move over the support material.
Chromatographic separations can be carried out using a variety of supports, including
immobilized silica on glass plates (thin layer chromatography), volatile gases (gas
the protein. If the protein has more positive charges than negative charges, it is said to be
a basic protein. If the negative charges are greater than the positive charges, the protein is
acidic. When the protein contains a predominance of ionic charges, it can be bound to a
support that carries the opposite charge. A basic protein, which is positively charged, will
bind to a support which is negatively charged. An acidic protein, which is negatively
charged, will bind to a positive support. The use of ion-exchange chromatography, then,
allows molecules to be separated based upon their charge. Families of molecules (acidics,
basics and neutrals) can be easily separated by this technique. This is perhaps the most
frequently used chromatographic technique used for protein purification.
Hydrophobic Interaction Chromatography ("HIC")
Not all of the common amino acids found in proteins are charged molecules. There are
some amino acids that contain hydrocarbon side-chains which are not charged and
therefore cannot be purified by the same principles involved in ion-exchange
chromatography. These hydrophobic ("water-hating") amino acids are usually buried
away in the inside of the protein as it folds into it's biologically active conformation.
However, there is usually some distribution of these hydrophobic residues on the surface
of the molecule. Since most of the hydrophobic groups are not on the surface, the use of
HIC allows a much greater selectivity than is observed for ion-exchange chromatography.
These hydrophobic amino acids can bind on a support which contains immobilized
hydrophobic groups. It should be noted that these HIC supports work by a "clustering"
effect; no covalent or ionic bonds are formed or shared when these molecules associate.
Gel-Filtration Chromatography
This technique separates proteins based on size and shape. The support for gel-filtration
chromatography are beads which contain holes, called "pores," of given sizes. Larger
molecules, which can't penetrate the pores, move around the beads and migrate through
the spaces which separate the beads faster than the smaller molecules, which may
penetrate the pores. This is the only chromatographic technique which does not involve
binding of the protein to a support.
Affinity Chromatography
This is the most powerful technique available to the chromatographer. It is the only
technique which can potentially allow a one-step purification of the target molecule. In
order to work, a specific ligand (a molecule which recognizes the target protein) must be
immobilized on a support in such a way that allows it to bind to the target molecule. A
classic example of this would be the use of an immobilized protein to capture it's receptor
(the reverse would also work). This technique has the potential to be used for the
purification of any protein, provided that a specific ligand is available. Ligand availability
and the cost of the specialized media are usually prohibitive at large-scale.
Other Methods
While the methods above are typically chosen for use in a purification process, there are
in fact many others that can be used. Each of these methods or techniques takes
advantage of a specific part of the protein being purified. The commonality is that all of
the techniques employed are based on the protein's structure.
Science Demonstration--Chromatography
Introduction
The purpose of the demonstration is to allow the students to get a "hands-on" opportunity
to do a scientific experiment. The focus of the experiment should be getting as many
students involved as possible, and to have fun with the experiment.
In the experiment, Blue Dextran is mixed with a dilute Phenol Red solution in a buffer
containing sodium chloride and sodium acetate at pH 5.0. At this pH, the phenol red turns
yellow (right in front of the students' eyes!). Upon mixing with Blue Dextran, the
solution turns green. Application of the solution to a Pharmacia PD-10 column separates
the green back into blue and yellow.
Experimental Procedure
1. Pre-equilibrate both PD-10 columns with at least 25 ml of equilibration buffer.
This allows the second column to be used while with first column is being reequilibrated.
2. Point out to the students beforehand that their eyes may play tricks on them. Let
them think that magic is at work here. Add 1-2 ul of Phenol Red to 500 ul of
equilibration buffer in a disposable 12 x 75 mm test tube. The solution turns
yellow.
3. Ask the students if they know what color is made by the mixing of blue and
yellow. Add 500 ul of Blue Dextran to the diluted Phenol Red solution to confirm
their guess.
4. Transfer the green solution to the PD-10 column. Point out that the colors are
separating from each other, but that only blue is visible at first.
5. Connect the powder funnel to the column. Add about 25 ml of equilibration buffer
to the funnel and watch for the color separation. The students will notice complete
separation of the blue from the yellow, with a white zone in between.
6. During chromatography, the students may have questions, and this is usually the
best time to answer them, as well as to explain what is going on. A simple
explanation of the theory is to compare the gel beads to "Wiffle Balls" and the
difficulty or ease that big and small objects may have in fitting through the holes
in the Wiffle Balls.