Weekend Protein Expression: A Surprising Success
Date: March 19, 2025
Last week in the lab, we faced a familiar problem: a protein requiring 4 hours of expression at 37°C,
but the OD600 only reached the induction target level of 0.8 late in the afternoon and
overall growth was very slow in M9 media. It was Friday, and none of us wanted to stay in the lab
until midnight. So, we tried something unconventional (to keep growth intentionally slow while still
allowing expression of our protein).
Instead of waiting, we made the following adjustments:
- Lowered the induction OD600 from 0.8 to 0.5.
- Reduced IPTG concentration from 1 mM to 0.1 mM.
- Switched the temperature from 37°C to 16°C.
- Allowed expression to run over the entire weekend (Friday 5 PM to Monday 10 AM).
On Monday morning, we were surprised to find the protein had expressed successfully! No visible
autolysis, no unpleasant odors (which often signal cell death), and the supernatant of the collected
pellets remained clear.
So, while not a huge observation, it makes life easier - this could be a game-changer for weekend
expression without needing to return on Saturday. If you're in a similar situation, give it a try!
First Entry: GST Removal at Low pH
Date: November 20, 2024
Welcome to my first entry! Here, I plan to share small observations that are too tiny for traditional
journal publications but might still prove useful to others. These posts are AI-generated but based
on my actual experimental notes, and I make sure to thoroughly check everything. Let's dive into the
first story:
Yesterday, I encountered an interesting challenge while removing GST from a solution buffered at pH
4.5 (sodium acetate buffer). Cytiva/GE advertises their resin as being compatible with low pH
conditions, meaning the resin won't degrade. However, I wasn't sure if the GST-GSH interaction would
remain intact at such a low pH, allowing me to effectively trap and remove GST from my sample.
To investigate, I started by looking into the pKa values of glutathione (GSH). GSH has four pKa
values, and none are near pH 4.5. Here's what I found:
- pK1: 2.12
- pK2: 3.53
- pK3: 8.66
- pK4: 9.12 (at 25°C)
You can see the pKa values for yourself here. Based on these values, GSH behaves similarly at pH 7 and pH 4.5.
To be thorough, I also examined the molecular structure and its interaction with GST. You can find a
detailed view of the GSH-GST complex here (zoom in on the glutathione molecule).
Despite these findings, I had my doubts. I even asked ChatGPT, which was skeptical about whether the
hydrogen bonding and other interactions would hold at pH 4.5. But the pKa values gave me enough
confidence to proceed with the experiment.
The result? Success! The GSH resin trapped GST proteins perfectly, even at pH 4.5 (even though the
GST formed white turbid aggregates in the solution!), and I could successfully proceed with my
GST-free protein. A small win, but one worth sharing. Hopefully, this observation helps others
considering similar experiments. Enjoy!