Did you check that it is in frame? Do you get any other small band maybe?

Hi there,

Did you check total on total protein extract? SDS/PAGE is not enough to state if there is expression or not (low level of expression will not be noticed if the POI migrates the same as an intense band of endogenous proteins). The difference between the 2 situations is of importance: if no expression it means there is either something wrong with the construct or the strategy for expression. If little level can be detected it might indicate the protein is not stable and degraded at a rate similar to the synthesis or if insoluble it might tell there is an issue with the protein folding (galactose induction is a strong induction and may lead to misfolding/mistrafficking). Have you got an other way to detect the protein like WB?

Dominique Liger. I haven't tried any other means for protein detection yet. Francisco Javier Alvarez, I am pretty sure about the construct but not the strategy as I followed the one suggested with the respective vector. There are bands in crude protein. But am not sure about the POI as am not getting any band after purification.

Hi, Alka. Have you tried to induce protein expression with the same reagents (ex. galactose) and obtained positive results? Are you running equal amounts of protein in the gels (after a protein assay)? I hope your problem gets fixed soon, I know how hard is to be stuck in such situations. Best wishes.

Thank you Francisco Javier Alvarez . I am not getting my Protein of interest by galactose induction although yeast internal proteins are there.

Can someone please suggest any good Ni-NTA-based purification kit for protein purification from yeast?

Regards

Alka Jangra

Thorsten Pfirrmann, I am using raffinose (2%) instead of glucose in maintenance media and raffinose (1%) + galactose (2%) for induction. Would that make any difference?? Do I need to change anything? Please suggest.

Hi Alka, I have developed an expression system in yeast for 6His and GST fusions (see Gene 399 (2007) 120–128) and my experience is that in minimal medium the yield is quite low and purification on Ni-beads is not very efficient (especially in yeast where lots of other proteins can compete with the matrix). Also, in your system the protein is secreted or is it cytoplasmic?

Hi Marco Geymonat, my protein of interest is cytoplasmic.

There are various parameters to take in account:

1) the medium used for induction: rich medium gives you much better yield but you cannot select for the plasmid (if you don't use my system :-) so the best is to make an o/n culture in minimal and then in the morning pellet the cells and re-suspend in rich + galactose (of course you need to avoid glucose in the o/n culture)

2) the amount of cells you collect and the efficiency of protein extraction. I use the fastprep machine with 0.5 mm glass beads. 4 bursts for 20 sec each.

3) the level of expression of your protein. If it is low the 6His method is not very good since you have lots of competition in the crude extract. Maybe you need to do a pre-purification with another column to enrich the content of your protein. Alternatively you add another tag (FLAG, GST...) that has less competition with yeast proteins. In my hands the best is MBP (but is a big tag).

Anyway, before any attempt of purification I would check with an anti 6His antibody (as suggested by Dom) if your induction has worked well and if it is worth proceeding.

Good luck.

Thank you Marco Geymonat. I used to grow cells o/n in minimal media+ 2% raffinose (instead of glucose) and in the morning, the cell pellet was transferred to media containing raffinose (1%) +galactose(2%). Should I need to replace raffinose with glucose or omit out raffinose from induction media?

Hi Alka ,

Primary culture: Set primary culture for over night in YNB media (2%glucose) with absence of your amino acid you use for selection.

Secondary culture: Use this culture to set large secondary culture in same media and grow for 24 hrs ( 24 hrs growth gives you good amount of cells and in the end the glucose will be used by cells so no glucose inhibitory impact. during induction step ).

Induction step: Now you induced these cells by adding 4X YPG media (yeast extract, peptone and galactose) to bring final volume 1X YPG ( 250 ml 4X YPG to 750 ml secondary culture this will gives you 1XYPG in 1 ltr) and grow cells for 16 hrs.

Good luck

the o/n culture in minimal medium to select your plasmid should contain raffinose 2% (I sometimes use 1% sucrose). The day after you pellet your cells and re-suspend them in YEP (rich medium)+ 2% galactose (no need of other C source) and induce for 4 to 6 h. Do not use dextrose (i.e. glucose) at any step (nor in the o/n culture nor during the induction) otherwise your induction with galactose will fail. It is not enough to consume all the glucose (btw 2% is in large excess so not sure you will consume all of it in minimal in 24h).

Hi Macro,

We use this protocol regular basis for successfully expressing protein in yeast .

24 hrs secondary culture growth work perfect to deplete the glucose and further inducing the protein by galactose in our case.

Best

Nitesh

Hi Nitesh,

I skip the minimal medium step because in my system the plasmid is stable and I can maintain the strain in rich medium. I cultivate only o/n in YEP+ 1% sucrose and then induce for 4 to 6 hours (by adding galactose in powder). In these conditions traces of dextrose impair the optimal induction.

Best

If the effect of glucose on induced expression is excluded and the protein still cannot express, it may be necessary to optimize the induction time and temperature.

Thank you everyone for your valuable suggestions.

Regards