6 replies to this topic

[ram]

We are trying to express a 66kD enzyme in pASK-IBA7 as described shortly below. The problem is that we are getting quite low levels of expression (Kindly have a look at the attached SDS-PAGE image. Lane 1: Desired protein extract. Lane 2: Protein extract of cells carrying empty vector). Does anybody suggest any modification in the conditions to get higher expression? Has anybody been able to get higher expression with some different conditions?

(1) Ligate the PCR product in pASK-IBA7 vector
(2) Transform in DH5alpha competent cells
(3) Pick a single colony and grow in 5ml LB media + Ampicilin for 5hours at 37C
(4) Transfer the 5ml culture to 100ml LB media + Ampicilin; grow at 37C till OD between 0.5 and 0.6 reaches
(5) Induce with anhydrotetracyclin (concentration as per manual), grow for 20hrs at 18C
(6) Cell harvesting and lysis

Thanks
Ram

[T C]

Try changing the expression strain....BL21?

[Thez]

No, DH5a -like top10 - is a cloning strain and cannot be used for expression... Use BL21, Rosetta etc

[ram]

What's the exact difference between cloning and expression vector? I have seen few papers (eg, this) where people have used Top10 for expressoin  

[Thez]

Hmm, maybe Im wrong?.. maybe DH5a and top10 cells can be used for expression. I guess it depends on the vector youre using. Im working with vectors containing the t7 promotor necassary for expression and DH5a and top10 cells dont have the gene coding for the t7 polymerase. Perhaps these cells contain other polymerases compatible with your vector?

This is (clearly:) not my area of expertise, and  I dont hope I brought to much confusion.

btw, some of the proteins we are working with show a higher expression when incubated 3-4 hrs at 37c postinduction vs ON at 18c.

Edited by Thez, 27 July 2009 - 03:15 AM.

[klinmed]

ram, on Jul 20 2009, 04:54 PM, said:

We are trying to express a 66kD enzyme in pASK-IBA7 as described shortly below. The problem is that we are getting quite low levels of expression (Kindly have a look at the attached SDS-PAGE image. Lane 1: Desired protein extract. Lane 2: Protein extract of cells carrying empty vector). Does anybody suggest any modification in the conditions to get higher expression? Has anybody been able to get higher expression with some different conditions?

(1) Ligate the PCR product in pASK-IBA7 vector
(2) Transform in DH5alpha competent cells
(3) Pick a single colony and grow in 5ml LB media + Ampicilin for 5hours at 37C
(4) Transfer the 5ml culture to 100ml LB media + Ampicilin; grow at 37C till OD between 0.5 and 0.6 reaches
(5) Induce with anhydrotetracyclin (concentration as per manual), grow for 20hrs at 18C
(6) Cell harvesting and lysis

Thanks
Ram

The vector pASK-IBL7 uses the tet promotor/operator. The producer of the vector (IBL) state "The tet promoter system is independent of the E. coli strain. The following strains were successfully tested: JM83, WK6, B, BL21, MG1655, W3110, XL1-Blue, BL21-
CodonPlusTM".

As for the T7 system, any E.coli background can be used PROVIDING it contains the DE3 lysogen (this contains the coding region for T7 polymerase).

BL21 has the advantage that it is protease deficient (Lon-, OmpA-).

Is the gel shown soluble protein or total E.coli extract (whole cell lysate)? If soluble you could have a folding problem.

I would certainly try different inducer concentrations/induction times. Only if the problem persists would I then move to "fancy" strains like Rosetta.

Hope this helps

[ram]

Thanks for the information Klinmed and Thez!
The gel picture is of crude bacterial lysate.