Time to get to our DHFR project. As we have explained before our aim is
to express, purify and do some characterization of DHFR while learning a few
things along the way.
In our previous posts, we discussed how to plan a
project, identify the key characteristics of your sample and target protein, along with reviewing the key analysis methods to be aware of. We also looked at the thinking around whether you should tag your protein or not and looked at
the key/common tags.
With that in mind, a couple of things we need to consider; since we are planning to characterize
DHFR using biophysical methods, we are aiming for milligram levels of active
protein at a purity of more than 95 %. For our purposes, it would make sense to
add a small tag, such as the Histidine tag (unlikely to interfere with our analysis methods and would simplify the
purification process). However, since we are doing this to learn and demonstrate
how to use protein expression, purification and analysis methodologies and the large variety of tools and techniques, we have (deep breath :) taken the decision to express and purify
human DHFR without a tag. Hopefully, this will not be too complicated.
Among the methodologies we plan to use, we have already discussed CIPP –
Capture, Intermediate Purification, and Polishing. Another methodology that we
plan to use throughout our project is Design of Experiments (DoE). DoE is a
structured approach to experimental planning that provides a framework to
explore parameters that may influence the outcome of your protocol. It will
help you minimize the number of experiments that you have to carry out, while
maximizing the information you get out of them in order to improve or optimize
your desired outcomes. As it should add logic and structure, we will try to apply
DoE to protein expression, purification and any other opportunities that we may
come across as our project progresses.
In our experiment, the first step is expression. To express DHFR,
we have chosen the most common host, E.
coli. There are many different systems that can be used (and we will review the pros and cons of the most common in a future post) but we chose E. coli as it is
simple, fast, reliable, low-cost, and easy to get high expression levels
(remember we need 95%). One drawback with this host is that there are no
post-translation modifications, such as glycosylation. With this choice of
host, there is also a chance that we will get our protein expressed in
inclusion bodies. While this would require having to refold the protein, it may
not be a negative thing for the purification, since the inclusion bodies
precipitate and therefore can be easily isolated, plus you
get very high purity of your target protein if it is expressed in IBs.
In our next post, we will look at the gene construct, vector and the
cloning of the DHFR expression vector in the E. coli host.
Meanwhile, if you
are interested in learning more about Design of Experiments for protein
expression and purification, you can take a look at our handbook.
