How can I increase protein expression in S. cerevisiae? - (Jun/18/2008 )

Hi everybody,
I am suffering for months to increase the level of my expressed protein, SESTRIN, from parasite S. mansoni. I cloned it in pYES 2.1 TOPO vector and transferrred in K601 S. cerevisiae cell line. I did 24 hrs galactose induction and crushed the cell by bead beater. From 400 ml starting culture I have 5 ml after final extraction. From that I loaded 12 ul ( I have not done any quantitation) in SDS gel and could not detect the protein. Then I did Western using His-tag antibody from sigma, I saw the single band of expected size ( 70 kd) but its very less in amount. I blocked the membrane o/n in 5% milk with Tween 20 and incubated 3 hrs at RT with the untibody. The detection is by BCIP/NBT.
Does any one have any suggestion so that I can increase the amount of my expressed protein?
Thanks,
Debalina

Hi,
Since I am having experience in expression of proteins in yeast I want to give some suggestions to u.
1) Lysis of the yeast cells by glass bead method, U will get very less protein. better to search another method to lyse the cell like Homoginization or sonication.
2) Use trace metals in expression media to increase the protein expression.
3) Do the brad ford before loading into the gel for westren blot and load about 100 to 150microgram of protein in each well or u can try eith differen concentrations.

For further questions please welcome.

bye all the best.

Hi Prabhu,
Thanx for the informatio.

1) Can you tell me the output or other specifications of the sonicator to use for cell crashing?
2) Which trace metal you will suggest and in what amount for a 1 lit expression medium. I am using SC medium which is 6.7 gm YNB / lit with amino acids, separately added. I do over night cell growing in this medium with 2 % glucose and then from that culture I dilute (OD600 =0.4 ) cell in the expression medium which is the same one except I add 2% galatose to induce the protein expression under GAL promotor.

Thanks again.
Debalina

1) Lysis of the yeast cells by glass bead method, U will get very less protein. better to search another method to lyse the cell like Homoginization or sonication.
2) Use trace metals in expression media to increase the protein expression.
3) Do the brad ford before loading into the gel for westren blot and load about 100 to 150microgram of protein in each well or u can try eith differen concentrations.

Hi,

1) Use 80% amplitude for 1 minute cycle (1minute process and cooling for a minute)(check the lysis through micrscope and optimize the cycles).
2) As u metioned in your reply you are using 6.7 grams of YNB for liter medium. But i feel thats its a huge amount. Try to use 0.17% of YNB (that means 1.7grams per liter. and use 0.5% of ammonium sulphate(5grams per liter) adjust the pH 5.6 to 5.8. add the aminiacid after sterile filteration (required).
3) When ever u want to take 2% glucose better to add in fed batch method that means first while inoculation add 1% and after 12hrs of growth add remaining 1%of glucose.
4)use the trace metals as fallow
Sol – A:

S.No Component Conc. (%) Quantity(10mL)
1 CuSO4 5H2O 0.3 30mg
2 MnSO4 4H2O 0.3 30mg
3 CoCl2 6H2O 0.05 5mg
4 ZnCl2 0.2 20mg
5 Boric acid 0.002 0.2mg

Sol – B:

S.No Component Conc. (%) Quantity(10mL)
1 FeSO4 2H2O 0.5 50mg

Add Solution - A 1% to the medium
Add Solution - B 0.6% (Do the sterile filteration of these two Trace metal solutions and add before inoculation)
5) Make sure that while inducing with galactose, glucose concentration should 0 or less than 0.2%

Regards
prabhu.