26 replies to this topic

[dreamcatcher]

Dear all,
                   I have tried to express my protein - sucrose synthase from rice, in [/I]P.pastoris. Althuogh I tried both electroporation and " EasyComp" from Invitrogen, I could not get overexpression of my protein.
Here are my inducing conditions:
                                                  30 C
                                                  150 rpm
                                                  medium BMGY FOR 16~18hr
                                                               change to BMMY OD600=1
                                                 used 0.5% methanol for induction  
                   I am curious that should I try higher methanol for induction or try to incube in different temperature?
Or how can I get an overexpression of my protein?

thanks

[celvas]

Hi,

you say you could not get overexpression but did you get any expression?

I am working too with Pichia p. and there are several factors which can affect the expression of your protein.

[dreamcatcher]

To celvas,

Yes, I did get expression of my protein, but the activity is quite low (specific activity=0.1~0.3, for crude protein).

Also it's hardly seen on SDS-PAGE and western blot.

And it is hard to purify it, if the activity is low. Cause I added stop coden before the His-tag. I was afraid that His-tag might affect protein activty.

Could you tell me how can I improve the expression of protein ?

thanks a lot for your advice : )

[celvas]

Hi again,

as you say you have poor expression of your protein since you have low level signal with western blot.

About the growing parameters I think 300 rpm is better than 150 rpm.

Also, some proteins are better expressed with higher amounts of metanol, try 1% methanol instead 0.5%.

Another interesting parameter is degradation of the recomb-protein. Did you see some amount of proteolysis in westblot?

Other possibilitys are related with plasmid. What kind of vector did you use? Is your protein secreted or not? Was your plasmid completely linearized before yeast electroporation?

About your purification problem I'm not sure if I have understood you. You say your encoding gene has a stop codon before HisTag. If so, you can not hope your protein has been produced with HisTag because protein codification finalizes with a stop codon. Therefore you can not use chromatographic techniques related with HisTag proteins.

Edited by celvas, 02 July 2004 - 02:28 AM.

[dreamcatcher]

celvas, on Jul 2 2004, 12:09 AM, said:

Another interesting parameter is degradation of the recomb-protein. Did you see some amount of proteolysis in westblot?

Thanks a lot !!!

I used pPICZA as plasmid for intracellular expression.

I'll change my protocol and see if any changes.
And I plan to use ion exchange for purification.

But about the "proteolysis in westblot", how can I see it?

[celvas]

Although we use pPICZalphaC vector for extracellular expression I think these parameters I have told you can affect your protein expression.

You can see protein degradation with western blot. If there are additional bands to those of interest they should be degradation products that compromise the yield of your r-protein.
If these band exist you must be sure they are not caused by western antibodies inespecificities; check this by loading sample controls with no r-protein expression.

Edited by celvas, 02 July 2004 - 08:33 AM.

[vadivu]

Respected Sir/Madam,

I am working in Pichia pastoris. I have reached till the expression.
I am using pPIC9 plasmid vector for my protein expression.
For expression, the clones are selected based on the high resistant to G418 and as per the literature high resistant means high copy number. I have gone upto 5mg/ml G418. And we have performed plate assay also to check for its activity , it shows positive.
Then i have taken those clones which are positive in assay for expression
i have checked the expression in YPD, BMMY media , now trying with YNB media . and also at different pH 5 and 6.
There is no expression, confirmed with western blot too.
i have even tried by adding protease inhibitor ..its not working
parameters for expression used are
incubated at 30 degree at 250 rpm.
so what you suggest me to do next to solve my problem?
Please do reply for my question
i will be thanfull to you if you can solve my problem.
Thanking you
vadivu

[celvas]

Hi,

I would like to help you but I work with other vector, pPICZalpha. I do not know the features of the pPIC9 vector, if it allows for extracellular expression or not, time of induction...

I suggest you to check the parameters that can affect your vector-dependent protein expression.

Good luck

Edited by celvas, 30 August 2004 - 03:12 AM.

[Gongzuo]

Hi, you all,
I am a new worker in this area.  I am trying to express a protein by using Pichia pastoris.  We got the expression kit from Invitrogen.  I already have my target gene cloned in pGAPZalphaB and also in pPICZalphaB.
Now my problem is that we cannot get yeast colonies on YPDS-zeocin plates after electroporation of the competent yeasts with the linerized gene construct.  I have discussed this problem with seveal professional people, but nobody can find where my problem is.  Nobody knows why.
Did anybody get this problem before?
How to make sure that our gene construct can be transfected into the yeast ?
Your answers will be appreciated.

Sam

[Naren]

Vadivu,

I have experienced the same problem with my protien. it does not express in shake flask culture in minimal media like YNB....... for couple of reasons....
Proteolysis in minimal media is to its highest level.......
Proteolysis in oxygen deficient media is to its highest level....

So if you try secreting them in fermentor with at least 30-50% saturation and moderate cell densities of 200 g/L (WCW) and go for induction that could solve your problem....

Secondly if you don't have fermentor.... then try to use some some stirle tubing and pump some air while using shake flask.... that might help you a bit....


Hope that might be of some help to you...

[Martorse]

dreamcatcher, on Jul 1 2004, 02:59 AM, said:

Dear all,
                   I have tried to express my protein - sucrose synthase from rice, in [/I]P.pastoris. Althuogh I tried both electroporation and " EasyComp" from Invitrogen, I could not get overexpression of my protein.
Here are my inducing conditions:
                                                  30 C
                                                  150 rpm
                                                  medium BMGY FOR 16~18hr
                                                               change to BMMY OD600=1
                                                 used 0.5% methanol for induction   
                   I am curious that should I try higher methanol for induction or try to incube in different temperature?
Or how can I get an overexpression of my protein?

thanks

Apparently protein expression in Pichia is quite sensitive to temperature, and stops above 32ºC. Invitrogen protocols strongly recommend to grow strains at 28ºC, to prevent overwarming in cultured flasks (be aware that even if temperature inside the shaker is 30ºC, the flask content can get warmer due to yeast growth). Wish you luck,
Marta

[Martorse]

I am trying to express a protein in Pichia pastoris. Problem is, I have noticed that growth of transformants in MGY (minimal medium) is really slow or even non-existing when I inoculate them after growth in YPDS. What can I do?

[clone]

HI,
anybody can tell me how much linearized DNA u use for electroporation transformation in Pichia.
and How much of  circular if any.
thanx

[tweete07]

Hello

It might be late for an answer but you can download the manual from the invitrogen web site it will tell you exactly how much linear DNA or circular DNA. Just follow what they say that's what I did to teach myself how to use pichia which doesn't I don't have problems but overall it works OK eventhough I wish I could improve my expression yields.

[gretafaccio]

Hi all,
i'm trying to express my flavoprotein in Pichia pastoris via pPicZalphaA cvector according to the invitrogen protocol but... where is my protein? i cannot see any band corresponding to it in SDS page and western blot!
growth conditions are the suggested ones ... no idea really... any brilliant advice for me?
many thanks by now and hope to read from you.