I have a current conventional purification protocol on GSH-sepharose beads that I need to improve yield around 2.5-3 mg/mL, but also reproducibility and larger scale production. For this I was thinking to test different parameters such as IPTG induction and expression at low temperature (17-20°C) O/N compared to the current 30°C 5h. Moreover do you think that comparing two lysis method such as sonication vs french press can be useful ? Or to increase the glutathione concentration during elution (10mM 20mM 50mM ?) and controlling a slow flow rate during binding? Finally what are the steps that will allow me to increase the yield and reproducibility towards large scale production ?
Thanks !
I am assuming you are expressing in E. coli?
Firstly, you can try different IPTG concentrations e.g. 0.2mM, 0.5mM, and 1.0mM - for protein quality purposes, I would recommend you let the cultures cool down at 4-8 degrees prior to adding IPTG, that way the protein folding occurs at a slow rate, and letting the expression go at 18 degrees over night.
Lysis should not have a ton of impact on yield as long as the lysis is complete and you do not let the suspension heat up too much. Do make sure to include protease inhibitor in your suspension.
After your elution, you can also just take 5ul of your beads and mix them with SDS-PAGE sample buffer to see how much of your protein is still stuck to them.
Aside from using even higher glutathione concentrations, you can also extend the binding time so that you ensure that the largest amount of protein sticks to the beads, and also let your last elution incubate for longer to wash off as much protein as possible.
Hope this helps for now.
Dear Dimitry
to answer to you question we need to see data to undertand which id the main step that limit your yield, the expression Level, the solubility, the partial binding in The purification?
Because different problems may require different Solutions.
i do not think that sonication and french press will result in huge different if their Protocol is correct and buffer the same.
to standardzie the growth and improve the bacteria biomass and therefore the volumetric productivity i suggests to try the Enpresso B growth media using induction at 25C for 24h.
you may found more information about this media and its protocol on my blog ProteoCool https://proteocool.blogspot.com/
Good luck
Manuele
Dear Dimitri,
1) Expression
Its usually a safer option to express protein overnight (16-20h) at low IPTG conc. (0.1 - 0.25mM) and low temperature (16-25*C). You also might want to test different expression stains (BL(DE3) RIL very often is a good choice).
2)Purification
There are few important considerations here. GST binds better to the beads at lower pH (6-7) and elutes better at higher pH (7.5 - 8.5). When you add reduced glutathione to the buffer (usually 10-20 mM) pH drops drastically. So always adjust pH after addition of the reduced glutathione. Low pH is the most common cause of inefficient elution. Of course all this must not collide with the pI of your protein: keep pH of the buffer at least 1 point away of the pI and never cross it during the purification. For instance, if pI is 7 use buffers in the range 8-8.5, or if pI is 9, buffers should be 6-9.
High NaCl/KCl may also help during the elution.
Good luck!
Anton
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