I induce 8 ml of over night protein culture that having kanamycin in 800ml LB agar and 800 ul kanamycin then incubated at 37C for 4 hours and 22C over Night induced with 800ul 1 M IPTG for 3 hours and harvest by centrifugation 15000 at 4C but I didn't get any protein when I purified and check in gel
you can increase the concentration of IPTG this might increase the protein concentration
I think anywhere between 1 to 0.5mM IPTG concentration would be fine, which is what you used. I would suggest changes in your protein purification protocol. I would suggest saving all the fractions starting from an uninduced cell culture, induced cell culture ( to check to see if there's any problem with IPTG induction), and collecting your Flow through and washes from your purification process and running all these samples on a gel to see if your protein might be in one of these fractions, due to efficient binding to your affinity column etc.
You can probably try spinning down your large scale culture at lower rpm(~5000).
Hi there,
Why do you grow first at 37°C then at 22°C (o/n?) before induction of 3h (is this actually what you do?)? What is actually the OD at which induction is performed? If you incubate 4h at 37°C then o/n at 22°C starting from a 1/100th dilution OD might be very high... For better expression, cell has to be actively growing so OD between 0.5 and 0.7 (exponential growing phase) is better. Furthermore If induction is done at 22°C, 3 hours sounds a bit short to me (I would go for at least 6h up to o/n induction). And prior to purification I would go for SDS PAGE analysis of the cell content (compare induced and non induced, compare total extract ans soluble extract).
Here your problem is lower expression or no expression at all?
If it is lower expression you can try the options mentioned below.
This works fine with us always.. at times when we tried to express big proteins, we had problem ..there we succeeded by reducing the culture shaking rpm as well as reducing temperature from 37 to 30,25 or even 20 we tried and we got good results.
If no expression at all, you have to review your entire expression experiment step by step. Like confirming the gene is in correct reading frame, no stop codons in reading frame, nature of your gene product ..like is there any chance of the product to get degraded by the host itself..or if it is toxic to the host, it may not allow the expression of the gene..size of the gene product etc etc..
I am dealing with two protein one I got very low expression and the other I didn't get an expression at all
I reduce the temperature from 37 to 22 just to check if i will get expression the OD 0.7
the induction in 22c was overnight
Hi again,
Have you considered trying other strains as a host like the ones encoding for rare tRNAs like Rosetta?
Hi Wafa,
Another suggestion that worked for me, is to try to do codon optimization. You may also try to grow your cells for more than 3h at 37C, I would take time points from 1h up to 5h after induction. Other suggestions is to grow your cultures at 18C or at room temperature overnight. Also, try different cells appropriate for toxic products (if this is your case). Have your check the pellet plus the supernatant by SDS-PAGE/WB?
When you fractionate your cell lysate, does your protein appear in the pellet or not at all? It is important to distinguish this because you can then differentiate whether it is a solubility question or an expression question. Generally, codon optimized, non-toxic proteins should express well at 37C, either being very soluble or being shuttled to inclusion bodies.
yes I transformed with BL21 and BL21 (DE3) strain but same results
I checked the cloning sequence by using Open frame reading but it is not contain his tag sequence is that could be the reasons?
hi wafa..i am afraid i am not getting the correct flow of your experiment..
usually what we do for recombinant expression is...
(for eg with pet32a vector and BL21 (DE3) host for expressing a eukaryotic protein)
clone the gene in to the expression vector.
sequence 2-3 randomly selected clones
analyse the sequence to confirm following things..if the gene is in the correct reading frame, position of start and stop codons, size of the gene product,any errors in the sequence, and whether the gene when translated gives the correct protein we need,
if everything seems ok we proceed with expression..
make a pre-inocualum (5-10ml) grow overnight at 37 , next day from this pre-inoculum, add appropriate amount , for example use 500ul to 500ml of fresh LB with antinbiotic ..allow to grow for 2-3hrs at 37 with proper shaking..
Then check the OD , if it is between 0.5-0.8 , the sample is ready to be induced.
Now we add IPTG (to a concentration of 1mM), allow to grow at 37 for another 4-5hrs. (you have to grow a flask of uninduced culture in the same way). Now remove a small aliquote (2ml ) of induced as well as uninduced sample for SDS-PAGE analysis. Then pellet down the rest of the sample in 50ml falcon tubes and store at -80.
First I will try the SDS-PAGE of the small aliquote first and confirm the presence of any new band in the induced sample compared to that of the uninduced sample. Then only I will proceed with the lysis of samples..
Selection of lysis buffer depends on which type of affinity column you are going to use.
.......
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