GST-Fusion protein and Biacore Analysis
Fun of Biacore
Biacore maker does not offer ready-to-run GSH chip. They do offer GST-Ab kit to capture GST-fusion protein, however, it has nothing to do with our routine purification with GSH-sepharose. Few people try to do it, the results are not great, we heard of that the Kd of GSH-GST fusion protein is around uM (e-6). With uM of Kd, how could you efficiently purify GST-fusion proteins from cell lysate? and run GST pull-down? Yah, something wrong here!
So, we try it. We tried to use amine coupling method to immobilize GSH
to CM-5 Chip, it does not work. It blocks the N-terminal that is
required for the binding of GST to GSH. We also tried to use carrier protein
through the -SH group of GSH, it does not work neither, it maybe
duo to the space hindrance. Finally, we try the epoxy activation (young
man, do not try it at home, it will destroy your Biacore!). By this, we
introduce an arm between GSH and matrix, this is exactly how GSH-sepharose
We inject 60ul of 0.2M GSH (pH8.5) over epoxy activated CM-5 chip over 30min, 500RU of GSH is immobilized. The chip has tested for the capacity, it can easily handle 1mg/ml GST-fusion protein.
Now, we are ready to analyze the interaction of GSH and GST fusion
protein. 5ul of GST fusion protein (0.3mg/ml, 5uM) is injected over this
home-made GSH-chip over 5min.
It is an ugly Biacore curve! There are two interaction phases: Fast-on/Fast-off (we are not interested in it, and it is estimated around uM of Kd) and slow-on/slow-off. The slow phase of interaction is calculated as 11.13nM of Kd. That is in line with our experience of GST fusion protein affinity purification practice.
What are take-home results? If the GST fusion protein in your
cell lysate is high (above 1mg/ml), you can run it through column, you
will get enough GST fusion protein to play around. Analysis indicates that
0.3mg/ml sample will double the binding over 0.03mg/ml sample. If the GST
fusion protein in the lysate is low (less than 0.1mg/ml), you better do
it in batch. For example, you may mix your lysate with GSH-Sepharose,
go home and play with your kids, let it rotate over night.
Thus you give it time to form stable complex (induce-fit! compared with
other interations, e.g. Ab/Ag , the slow-on is 10 fold slower). Biacore
analysis shows that 5ul of sample over 5min will give you 4 fold binding
over 1min of injection.