I used NcoI and XhoI for the recombinant protein construction in pET28, and I found it had no any frame shift when simulate construction map in SnapGene software.
The problem is I always cannot get overexpessed protein after IPTG induction, not just one protien but all of them.
In case there is any issue about codon usage bias (my target gene is not from E. coli, my expression host is BL21, and the induction concentration of IPTG was from 0.5mM to 1mM), the prediction of relative codon bias scores had no significance difference.
And I pretty sure my targets are not toxic proteins.
So, Does anyone have experience on this problem, thank you.
Does your insert contains rare codons for expression in E coli ? If so use a E coli Roseta strain or equivalent
Yup, definitely sequence-confirmed.
Dear Alban, thanks for your recommendation. Only few of the sequences (less than 5 codon) were poor usage, but Roseta might be an alternative choice to try.
I cloned three constructs in pET-28 vector, none of them expressed in Bl21-DE3, but all of them over expressed very well in Rosetta-2DE3 with 0.4mM IPTG concentration (include both Kanamycin and Chloramphenicol).
Also, check if your protein is over expressing,, but is going in inclusion bodies instead.
Is your target protein his-tagged? If yes, you should run some western blots to confirm the expression.
Sometimes lowering the expression temperature under 36°C helps. A broad IPTG concentration range should be tested.
@I. C. Bustamante-Filho, yes, my target proteins are His0tagged and I the western blot with 6xhis antibody showed no expression pattern after IPTG induction.
Since none of them expressed, I assume that the condition must went something wrong.
@Bhanu P Jagilinki, do you mean I have to add both Kanamycin and Chloramphenicol in culture medium? What's the concentration would be? Is that concentration also suitable for transformation plating?
Thank you all.
pET-28 has T7 promotor but BL 21 cells don't have T7 RNA polymerase. You need to use a strain that has DE3 in its name, such as BL21(DE3), or as Bhanu suggested, Rosetta-2DE3.
Thanks @Jana, sorry about the incomplete information, indeed my expression host is BL21 Star™(DE3) and C43 (DE3). So it could not be the problem when use pET system to control protein expression.
Yes, u have got to add both the antibiotics in the culture if u r going to use Rosetta 2DE3 for ur pET-28 constructs. So, make these two stocks:
- 50mg/ml Kanamycin in distilled water
- 34mg/ml Cholramphenicol in Mol Bio grade Alcohol or isopropanol
Add these both stocks in 1:1000 dilution,, i.e 1ml of each of antibiotics in 1 liter medium.
From Transformation plate, starter culture and till dilution in larger media,, u always have to add both the antibiotics in 1:1000 ratio from the above mentioned stocks.
I would try varying a few parameters and seeing if you improve your results:
Thanks, Roger.
Those were great suggestions, I'll try if I have such materials.
Hi Vincent!!
Did you go through with your gene orientation. I mean to say that sometime the orientation of the multiple cloning sites is different for some vectors. Some having clockwise and some anticlockwise orientation. You need to check properly. If your gene ligated in wrong orientation to MCS then stop codon will come at first and ultimately you will not get your target protein.
If above mentioned points is fine in your case then there are several methods to get or enhance the protein expression. What do you mean here "The problem is I always cannot get overexpessed protein after IPTG induction, not just one protein but all of them." Can you clear this point. I can help you to enhance your protein expression...
Good Luck.
Hi Gaurav Chhetri,
The orientation of the target genes was confirmed by sequencing.
My concern is the recombinant proteins cannot be overexpressed and sometimes no any target protein be found. Thanks for asking.
Are you sure you are not getting any expression? It could be that the proteins are insoluble, you can check by western blot of the whole cell extract after lysis in SDS-sample buffer. If that is the problem then the suggestions by Roger bradley Dodd may help.
Also you only said you checked for frame shift with genesnap, if you didn't sequence the plasmid you need to.
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