Dear All, I am trying to express and purify a protein of MW 220 kDa (full length). It has an extra GST tag in it. The strain I am using is Rossetta (so issue of rare codon is ruled out). Now, when I am trying to express this protein, its yield is very very low. It shows lots of degradation as well. For that, I used PMSF 2 mM but issue remained same. Condition is 0.3 mM IPTG induction at O.D 0.6 and then overnight shaking with 150 rpm at 20 degree. Anything which I can do to improve the yield and avoid degradation. Looking forwards for your suggestions.
Thank you, With kind regards
P.P
You can use insect cells as suggested. If you dont have them then you will have to lot of troubleshooting. You can vary iptg conc, induction time, expression time, temperature. Also pmsf is not stable for long time, you can also use protease inhibitior tab. I expressed protein double of your size, and it took lot of efforts to optimize it.
I suggest that you induce your expression at a much later phase of cultivation. As a first step I would follow the growth rate curve with and without IPTG induction. Then you can determine if your protein cause a metabolic burden on your cells. You can simply see if the cells starts to grow slower after IPTG addition. In the second step you add IPTG at a late logarithmic growth state. You also add different concentrations of IPTG, up to 10-fold more than you have inducated.
Best of luck
Mahesh Chandak So can you please suggest me the conditions in which you successfully achieved the good expression of your protein ? It would be a great help. Thank you
Identify your final OD without IPTG addition. In the next experiment you add IPTG at 80-90% of your final OD. Add up to 3 mM IPTG, but vary the conc. Wait 1-3 hours and follow the expression levels by SDS-PAGE and then you can determine the optimum parameters.
Dear Prem
proteins higher than 200Kda are certanilly challenging exprcially in e.
coli and yoy cannot expect amazing yields.
for e.coli i suggest to you to possible appoaches:
1) Use an high cell density media as Enpresso B, growth on at 30C abd then induce 24h at 25C in this way probably you will not solve the degradation problem but will allow to you to imprive the volumetria protein yields of about 10-20 times (mg/l)
you can find more information about high cell density and Enpresso on my blog: ProteoCool ( https://proteocool.blogspot.com/?m=1) at Page2 ProteoCool n°5 and ProteoCool n°7.
One other possibility ( but expensive) is order to Genescript or Geneart a sintetic dna codon optimized for expression in e.coli. In this way you can use standard bl21 instead rosetta and is it possible that this will help to you to reduce degradation and improve productivity.
Least but not last, recently i expressed a 175kda protein, cysteine rich with Expi293 mammalian expression sistem.
https://www.thermofisher.com/order/catalog/product/A14635
it is also expensive and you need a shaker incubator with CO2 8% but it is very powerfull and simple to use and set-up if you have the instruments . the protein directly secreted into the media (yield about 10-20 mg/l) and with just 1 Imac step i was able to obtain very high purity level.
good luck
Manuele
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