1. How to scale up the expression of fusion proteins?
2. How to improve the purity of fusion protein to 90%? I have tried affinity chromatography, but the purity by SDS-PAGE was not satisfied.
3. Besides calcium phosphate transfection, are there any other transfection methods recommended?
It would be nice if anyone could also give me some related references. Thank you very much.
It is all protein dependent! However, in S2 cell, I have always used calcium phosphate for transfection and selected for stable cell lines with puromycin in complete media containing FBS (10% serum) in a T25 flask. Then gradually scaled up to T75 with puromycin selected. At some point, you can start changing the media gradually to reduce the FBS (albumin) down (5% FBS, 2.5% FBS etc.). Then scale up using T225 flask and then move up to 150 mL, 500 mL and 1000 mL flasks but with shaking - now when you get to this stage you can decide how many 1000 mL you would like for the expression...For induction copper sulphate ( 0.5 - 1 mM) was always used... Useful to do a trial expression and scale up accordingly for targeted yield..
Tips:
1) MBP tag can provide solubility!
2) The pMT-PURO expression vector was used!
3) Induce cells around 6mill/mL and leave for 7 days for expression under shaking (110 rpm) or do a trial expression to find out when to stop expression by taking samples on days 1, 3, 5 and 7 for SDS-PAGE gel and western blotting.
4) For purification - spin media and filter with 0.22 micron filter followed by Ion exchange chromatography ( Capto-Q or Capto-S) followed by affinity chromatography ( MBP Column, Ni excel column fusion has his tag etc.). And polish with gel filtration as a final step.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2553637/
Thanks, Bubacarr G Kaira ! I still have some other questions.
1. When you shaking, did you add Pluronic F-68？
2. If I have selected stable cell lines, do I need to keep puromycin in the medium all the time, even during the expression of fusion proteins?
3. The plasmid used in the transfection is disolved in ddH2O or buffer provided?
4. Do you have some other medium recommended besides Schneider's Drosophila Medium(Invitrogen 21720024)?
Hi Xin,
For your questions -
1) I have never added Pluronic F-68 while shaking.
2)The stable cell line selection with puromycin can be stopped when you perhaps start to reduce the 10% FBS with an ExpressFive media.. Before changing media take some stable cell lines out to store for future use (you need complete and incomplete media with added DMSO for storage - check literature for more info)
3) For me I usually perform a maxiprep followed by DNA precipitation - now during the DNA precipitation at the last step when you do Ethanol step - air dry in the hood ( Sterile) and use MQ water or autoclaved water - do all of this in the hood..
4) I have always used the complex Schneider Drosophila S2 medium with added FBS (10%)...The ExpressFive medium can be used to reduce the serum and continue using the ExpressFive for protein expression...
You can also add antibiotics but must add L-Glutamine medium ( double check online)
Hope this helps..
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