Have you checked for hGM-CSF in the intracellular fraction?

I checked but i also saw no difference comparing to negative control.

I also checked recombinant vector, strains, all are ok :(

So the sequence of the vector is OK and there is no expression in either the extracellular or intracellular fractions. How are you checking for expression?

Did you check for premature termination and internal initiation? You may check this using high titer polyclonal antibodies against the target protein on western blot. Something very strange. The vector is supposed to be a E.coli-yeast shuttle vector!

Do you add antibiotic (G418) to your cultures to select for the plasmid? If protein expression is toxic, you will lose the plasmid pretty fast otherwise.

Did you DNA sequence the vector to see whether your insert is in frame with the promoter? Did you induce for the expression?

It sounds like you may have a problem with your DNA sequence or you have a toxicity problem. Have you checked to see whether your protein has been expressed in yeast before? If it has, check your DNA sequence as per the comments above. If it hasn't you might consider an inducible system.

Dear all,

Thank you so much for your comments.

I checked DNA sequence- it's Ok. And this protein isn't expressed in the host cell (checked).

To check the expression: I did SDS-PAGE cell extract, supernatant (with and without precipitation-TCA) for 20 candidates and negative control.

I just added G418 to preculture.

And this expression system (TEF promoter) using glucose in medium as inducer

However I've not thought about toxicity problem. If that's the way it is, it means that there's no way to express this protein using this system!?

@Ravi Pottathil:You mean there are somethings wrong with target RNA transcription which directs target protein to misfolded form or blocking transportation? I just wonder, if so it must be have the difference comparing to negative control in SDS-PAGE gel.

Another question: Have you optimized your gene for yeast codon usage?

Pham, the TEF promoter is constitutive, so it will be on all the time. It may be that you need to switch to an inducible system. If you have a toxic product, that is one way to address the problem. I suggest you search to see what has done before. For example, I have just found this article that suggests toxicity may be an issue: http://www.ncbi.nlm.nih.gov/pubmed/22627758. There are plenty of others out there that will give you some ideas. Good luck!

If expression is low (and even if it is relatively high), it's unlikely that you will be able to detect the protein by visual comparison of cell extracts; this is not E. coli. Concentrated culture supernatants may be another story, but still it would be better to do a Western blot if possible, because hGM-CSF will be glycosylated upon entering the secretory pathway and will appear as a smear, rather than a band.

BTW, what secretory signals did you attach to the gene?

what I meant was that due to inappropriate codon usage one can end up with premature termination. This will show up in western blot. Do you have specific antibodies available to you? I have seen similar situation when we expressed HIV clones in bacteria.

Dear all, do you have any ideas about the protocol for expression, for example: innoculation, temperature, shaking speed, time to get sample...

@Tom Bender: Yes, i did

@Roslyn Bill: I succeeded to express this protein using inducible AOX1 promoter in Pichia pastoris. The secreted hGM-CSF migrated as a band at about 20 kDa, a diffuse band at the range of 29 to 35 kDa, indicating differentially glycosylated forms, and a band at 14,7kDa which is a non-glycosylated form on SDS-PAGE gel.

@Alejandro Martin: I used alpha-mating factor secrectory signal.

@Ravi Pottahil: I will do Western blot first to check again whether or not this protein is expressed.

Thank you very much!

Dear Pham, It sounds like you have solved your problem in P. pastoris. Why not stick with that yeast species? The glycosylation pattern is also likely to be less hypermannosylated in P. pastoris. If you want to work in S. cerevisiae, there are lots of inducible promoters that would be worth a try.

Thanks Roslyn,

My laboratory (in Ho Chi Minh, Vietnam) is not strong about S. cerevisiae, we just have some strains and some vectors (TEF and CYC promoter). And there are some reasons, We can't buy the others :)

I am trying to working in S. cerevisiae because:

(1) I want to choose the best expression system.

(2) The comercial hGM-CSF was produced by using S. cerevisiae

(3) Using P. pastoris with AOX1 promoter, methanol is the inducer - It's still not the best way to produce a drug

Dear Pham,

Have a look at this: http://tools.lifetechnologies.com/content/sfs/brochures/710_021524_pyes_bro.pdf and this: http://2008.igem.org/wiki/images/2/27/JHU_0708_paper_ForeignGeneExpression.pdf to give you some ideas.

There are some constitutive, methanol-independent systems (GAP) that can be used in P. pastoris as well. If you browse the web or PubMed you will find them.

Very best of luck,

Roslyn

@Roslyn Bill: Many thanks to you!