protein isolation from pQE30

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ggkk
ggkk's picture
protein isolation from pQE30

hi all,

I am trying to express protein in PQE30(qiagen). there is no desired protein at all. i used a protocol which involved washes with 0.5M Nacl+2% triton X 100 ,Washes with 0.5mNacl, followed with sterile water. is this the correct method?  I repeated this for thrice same result.
i am frustated with this. can anybody suggest me what to do?
thanks in advance.

Chin Fen Teo
Chin Fen Teo's picture
 Hi ggkk,

 Hi ggkk,

1. Have to try to perform a western (pQE30 should have a His-tag, if you didn't kill the tag during the cloning) to examine the expression, or you just did a gel stain? Not all the protein overexpression can be seen on the gel stain...

2. The washing steps that you mentioned, I assume it is after harvesting? I don't think that is the cause of your problem. You should try to vary your IPTG induction step (eg. IPTG concentration, time, temperature).

Good luck.

ggkk
ggkk's picture
protocol which involved

protocol which involved
 induction-----harvesting ------- incubation with lysis buffer for 4 hours--------soniction-----centrifugation ------washes of pellet with 0.5M Nacl+2% triton X 100 ,Washes with 0.5mNacl, followed with sterile water----solubilisation of pellet in 50mm carbonate buffer ,ph10-----collect supernatent after 2hr incubation at 37 degrees
i can't go for western blottting. suggest me a method that i can find my band on sds- gel .

Chin Fen Teo
Chin Fen Teo's picture
 ggkk,

 ggkk,

If you don't see the protein expression when you are running whole lysate with or without IPTG induction, western is the only alternative to tell whether your protein is there or not... Unless you are working with an enzyme which is not robustly expressed by the bacterial and you can easily assay for the activity.

And also, are you working with insoluble protein? I can't really tell, because the first half of your protocol looks like you are working with insoluble protein, but the second half of the protocol says otherwise.

Lastly, if it is a protocol established for the protein you are working with, make sure you add protease inhibitor and keep everything ice-cold because it is an awfully long procedure... You have to make sure that proteases are not degrading your protein during the process.

Again, if you don't get anything at the very beginning, then you have to go back to optimize your induction condition.
 
Anyway, that is what I could think of.

Good luck.

kjosephs
kjosephs's picture
What host strain are you

What host strain are you using?  IPTG inducable expression from pQE30 requires that the lac repressor be supplied in trans, typically from the pREP4 plasmid.   See QIAexpressionist pages 15-17 for details.
 
www1.qiagen.com/literature/handbooks/literature.aspx

Also the protocol you described seems a bit extreme in terms of long incubation times, pH, and temp.  Is this an established protocol optimized for your protein?

ggkk
ggkk's picture
hai all,

hai all,
 i can't go for western. the washing steps procedure was estabilished for pEt29 vector. i used the same here. but no protein.
when i checked cell lysates ( harvested cells + lysis buffer+ sonicaton), i find the 130kd band in uninduced , induced ones.still, there is no change in intensity of band.
i have used  Xl-blue cells as host.
can any one suggest  a method to  get protein.
thank you

Neznika
Neznika's picture
Did you check IB on SDS-PAGE?

Did you check IB on SDS-PAGE?

ggkk
ggkk's picture
what is IB?

what is IB?

Neznika
Neznika's picture
IB - inclusion bodies. When

IB - inclusion bodies. When you separated lysate by spinning after a sonication you have two phases - there are liquid and solid. Liquid phase mostly includs is the soluble proteins, lipids, nucleotides etc. ( it is the supernatant). The solid one is cells walls, different compartments of cells, nucleotides, unsoluble proteins  and etc.(it is inclusion bodies, look for details in wikipedia for example). So did you check IB on SDS-PAGE? If YES - how did you prepare a sample for electrophoresis?

ggkk
ggkk's picture
yes, i checked inclusion

yes, i checked inclusion bodies  on sds page. after centrifugation, the pellet was addede with 5 ml of lysis buffer . that viscous samople i loaded in sds-page.  but the picture was so bad. entirely the lane showed smear. thats why i went for the three washes protocol which i use for pet vector system. but there is nothing on page.
 

Neznika
Neznika's picture
OK. I got it. Looks like you

OK. I got it. Looks like you have check three first common problems:
1. No expression
2. Proteolysis/autoproteolysis
3. Solubility
As I know about your protein nothing and you had not find out your protein in a supernatant after lysis, let try to improve the lysis step for IB. This can allow to check the questions 1,3. So:
a. Take a part of IB and dissolve at 8M urea (about 1V IB : 10V urea; 100ul -1ml will be enougth; Vortex couple minuts).
b. keep benchtop approximately 2h at RT, vortex again.
c. spin on a benchtop centrifuge 10min/ 13-15K rpm Note: if you will not get visually separated super - repeat this step.
d. Take aliquots of super from top level of liquid for SDS-PAGE.
e. Prepare at least 3 samples from these aliquots:
- 1V samples : 1V loading buffer
- 1V deluted 1:4 sample with 8M urea : 1V LB
- 1V deluted 1:8 sample with 8M urea : 1V LB
f. SDS-PAGE
The picture could be more informative and you can choose next step

ggkk
ggkk's picture
thank you. i will try this

thank you.
i will try this

Neznika
Neznika's picture
Don't pour a supernatant.

Don't pour a supernatant. Precipitate proteins with ammonium sulfate, spin, discard super and freeze a pellet at -20 or -80.

Vikush
Vikush's picture
Hi,

Hi,
You are writing that you are trying to express the protein in pQE vector in Xl1Blue cells. Am I right? Try to use M15 (pREP4) host strain, then you will get your protein.
Good luck

ggkk
ggkk's picture
means xlblue don't work for

means xlblue don't work for protein expression?