Lack of Expression in pQE30 clone - (Jul/17/2008 )
I have finally cloned my codon optimised DNA sequence into the SphI and HindIII restriction sites into pQE30 expression vector by Qiagen. The sequence is in frame upon sequencing and I have transformed into M15 and JM109 E.coli for expression purposes.
I have also posted a question that was answered by a Andriy regarding my clone on the Molecular cloning forum. He has mentioned a query about using SphI in which my supervisor has said would not pose a problem.
But upon preliminary expression in JM109 and M15, there were no intense expressed band seen as the protein profiles were similar to that of the controls used which were the strains that were transformed pQE30 vector into it0. I used 1mM and induced expresison at OD600nm 0.6.
I then repeated expression in both the strains by lowering the IPTG concentration to 0.5mM and the profile seems to be the same.
Should I increase my IPTG concentration to 5mM as some suggested or perhaps lower further to 0.005mM? I was also told by my senior to decrease the growth temperature to 25 degrees Celcius. But I thought it is only for solubility purposes?
I was thinking of detecting with Western blot but no 6xHis antibody...
I have also posted a question that was answered by a Andriy regarding my clone on the Molecular cloning forum. He has mentioned a query about using SphI in which my supervisor has said would not pose a problem.
But upon preliminary expression in JM109 and M15, there were no intense expressed band seen as the protein profiles were similar to that of the controls used which were the strains that were transformed pQE30 vector into it0. I used 1mM and induced expresison at OD600nm 0.6.
I then repeated expression in both the strains by lowering the IPTG concentration to 0.5mM and the profile seems to be the same.
Should I increase my IPTG concentration to 5mM as some suggested or perhaps lower further to 0.005mM? I was also told by my senior to decrease the growth temperature to 25 degrees Celcius. But I thought it is only for solubility purposes?
I was thinking of detecting with Western blot but no 6xHis antibody...
This is the expression profile of the total cell lysate
As highlighted in green are p11, highlighted in red are p14 and yellow or c(0) or c(3) are controls.
Those with [] brackets are 1mL fractions.
Other than the [], cells were normalised to T=0 hr.
All cultures were induced expression at OD600nm = 0.7.
Cultures were grown at 250rpm, 37 degrees Celcius.
I first tried with 1mM IPTG and then repeated it again by lowering the IPTG level to 0.5mM thinking there would be some changes but the profiles seem similar.
Hi
i can tell u that not all proteins can be see in commasie as induced compare to not induced ones.
As u said, if u r not sure, the best thing is to use W.B with anti His tag Ab.
UR induction profile seems ok (don't change IPTG conc.). U also should longer induction time 6h or over night induction.
good luck
Hi Amtash,
Thank you for your reply.
I am pretty new in this.
Not all proteins can be seen in Coomasie? Would you suggest I do Silver?
May I ask about IPTG? If I do increase it, would it make a difference? I did try increasing it but the profile is exactly the same! IPTG is not quantitative is it?
I will try overnight today and see whether I get something.
Thank you very much =)
Best,
Platyhelminthes
Hi Platyhelminthes
What i meant when i say that "Not all proteins can be seen in Coomasie" is that its sometimes difficult to see and identify
clear band of induced protein when u compare it to non-induced fraction (because of contaminating proteins or just
protein smear). As i said, the best way to be sure, is to do western blot (don't forget positive control), i don't know how
much silver can help u.
Concerning the IPTG concentration- 1mM is acceptable as "enough concentration" for induction. I don't think that
increasing the IPTG would help u.
What u can change is : 1) Induction OD- 0.8-1 instead of 0.6 2)which buffer do u use to test ur samples-fractions 3)do u
load enough culture (total proteins) onto agarose gel? 4) do u take OD in the end of induction (check that the cells are
not dead- toxic protein ?)
by the way JM109 are not expression cells (as much as i know) they use as replication cells for plasmids.
BYE
What i meant when i say that "Not all proteins can be seen in Coomasie" is that its sometimes difficult to see and identify
clear band of induced protein when u compare it to non-induced fraction (because of contaminating proteins or just
protein smear). As i said, the best way to be sure, is to do western blot (don't forget positive control), i don't know how
much silver can help u.
Concerning the IPTG concentration- 1mM is acceptable as "enough concentration" for induction. I don't think that
increasing the IPTG would help u.
What u can change is : 1) Induction OD- 0.8-1 instead of 0.6 2)which buffer do u use to test ur samples-fractions 3)do u
load enough culture (total proteins) onto agarose gel? 4) do u take OD in the end of induction (check that the cells are
not dead- toxic protein ?)
by the way JM109 are not expression cells (as much as i know) they use as replication cells for plasmids.
BYE
Hi Amtash,
I would also want to do Western to check. My problem is that I cannot identify the induced protein.
Thank you for clarifying on the IPTG concentration. I truly appreciate your input thus far. You have truly been very helpful =)
1) I have changed the induction today and there is still no difference.
2) I use the SDS-PAGE 1x dissociation buffer to the washed cell pellet and load 14uL into each well.
3) As above. I normalise the cells to that of T=o hr of induction because my seniors had problems answering the protein content query in their Viva's. Ergo, my supervisor suggested me to do so as a reference to 'single' cell production.
4) I take OD at every hour in order to normalise the cells. Reaches stationary phase with no unstable growth.
I use JM109 to compare the expression and protein profile to be written in my thesis the difference between using a cloning host cell and expression host cell to 'thicken' up my discussion.
In regards to your previous suggestion, I have taken OD and sample at T=6 hours and tomorrow morning at T=20 hours for the lab is closed in between. Considering Ampicillin is unstable, would my protein profile be okay? I am also wondering, considering the insert is codon optimised for expression, my clone is rather useless considering it hardly expresses and if it does after such long hours, right?
Best,
Platyhelminthes
What i meant when i say that "Not all proteins can be seen in Coomasie" is that its sometimes difficult to see and identify
clear band of induced protein when u compare it to non-induced fraction (because of contaminating proteins or just
protein smear). As i said, the best way to be sure, is to do western blot (don't forget positive control), i don't know how
much silver can help u.
Concerning the IPTG concentration- 1mM is acceptable as "enough concentration" for induction. I don't think that
increasing the IPTG would help u.
What u can change is : 1) Induction OD- 0.8-1 instead of 0.6 2)which buffer do u use to test ur samples-fractions 3)do u
load enough culture (total proteins) onto agarose gel? 4) do u take OD in the end of induction (check that the cells are
not dead- toxic protein ?)
by the way JM109 are not expression cells (as much as i know) they use as replication cells for plasmids.
BYE
Hi Amtash,
I would also want to do Western to check. My problem is that I cannot identify the induced protein.
Thank you for clarifying on the IPTG concentration. I truly appreciate your input thus far. You have truly been very helpful =)
1) I have changed the induction today and there is still no difference.
2) I use the SDS-PAGE 1x dissociation buffer to the washed cell pellet and load 14uL into each well.
3) As above. I normalise the cells to that of T=o hr of induction because my seniors had problems answering the protein content query in their Viva's. Ergo, my supervisor suggested me to do so as a reference to 'single' cell production.
4) I take OD at every hour in order to normalise the cells. Reaches stationary phase with no unstable growth.
I use JM109 to compare the expression and protein profile to be written in my thesis the difference between using a cloning host cell and expression host cell to 'thicken' up my discussion.
In regards to your previous suggestion, I have taken OD and sample at T=6 hours and tomorrow morning at T=20 hours for the lab is closed in between. Considering Ampicillin is unstable, would my protein profile be okay? I am also wondering, considering the insert is codon optimised for expression, my clone is rather useless considering it hardly expresses and if it does after such long hours, right?
Best,
Platyhelminthes
I now have expression =)
BUT, why is the protein larger than my expected band. It is around 20 kDa whereas my protein should be 18 kDa and the 6x His tag protein is 0.84kDa.
Does anyone have any clue?
Hi
Dont worry, be happy...
If u ask anyone, they will say to u that u "always" get ur protein not in the expected size (usually its few kDa larger)
u can run simultaneously two proteins ladder and u see difference in their running
Congratulation for ur expression, remember to treasure this moment.
bye
Dont worry, be happy...
If u ask anyone, they will say to u that u "always" get ur protein not in the expected size (usually its few kDa larger)
u can run simultaneously two proteins ladder and u see difference in their running
Congratulation for ur expression, remember to treasure this moment.
bye
Okay. Heehee Thank you sooo much again, Amtash.
Is there a particular reason why does it express slightly larger? E.coli has low post-translational modifications if not close to none right?
I did exactly that, I ran a 7 - 175kDa prestained marker along with my 2 - 212kDa marker today. I will check and see again..
Thank you. It is really worth it for I have been trying for 8 months to clone and a month to express and this is just my undergrad research project =s
I've attached the gel photo here where M is NEB's 2 - 212kDa protein marker and the second from bottom band is the 20kDa band. lanes 1 -3 are cell lysate from M15 cells whereas the remainders are the JM109 cells. All are from hour 6 - 7 upon induction