Expected protein size ? - fusion protein-size in kDa? (Jan/24/2006 )

Hi

I made some constructs where I have the gene I am interested cloned in frame with another gene.
I have 2 constructs :

46a.a. encoding gene + gene of interest
147a.a encoding gene + gene of interest

Because I wanted to check if the fusion proteins were being expressed,
I transfected cells with those constructs and run a western blot .


MY QUESTION IS:
Do you know about any program where I can type the a.a. sequence of the fusion proteins and get the expected weight in kDa?


I ask this because the results are a bit strange so, I am wondering if not only size but protein conformation affects protein run and therefore size?

Thanks

---

this should do the trick:
http://us.expasy.org/tools/protparam.html

the conformation should not be a factor if you are running a denaturing gel (SDS-PAGE). in these conditions, theoretically, the protein should be roughly linear with an overall negative charge. in reality, sometimes proteins may run a little larger or smaller than their calculated MW.


hope that helps!

---

Hi chempilot,

I will try this website.
But whata does it mean if I have 2 specific bands representing the same protein but with different sizes. Is my protein truncated? I really did't expect that because i cloned the gene in frame and checked a.a. sequence, there is no stop codon.

---

glycosylations? Is it bigger than expected?
it has already been disscussed in the forum. if you want to calculate the relative molecular weight, it is better to use non stained protein markers because the dye may change the migration profile.

(I experience it with two different markers. the difference you obtain is amazing)

---

Hi chempilot:

I went to the expasy website that you recommended.
It a great link to analyse proteins. Thank you very much!!!!!!!!!
The protein that is fused with another 147 a.a. at the N-terminal part is predicted to be 37.5kDa BUT is UNSTABLE!
That might explain why I see in my western not one but 2 bands. One of 37kDa and another of 27 kDa.

---------------------------------------------------------------------------------------
Estimated half-life:
The N-terminal of the sequence considered is M (Met).
The estimated half-life is: 30 hours (mammalian reticulocytes, in vitro).
>20 hours (yeast, in vivo).
>10 hours (Escherichia coli, in vivo).

Instability index:
The instability index (II) is computed to be 44.54
This classifies the protein as unstable.
--------------------------------------------------------------------------------------------
What do you think?
It might be because I cloned the gene with the start codon/ATG. maybe I should't right?

please tell me what do you think.

---

i have had mixed results with the instability index, so i wouldn't live by that prediction.

what is the fusion protein you are using: GST, thioredoxin, MBP? are the antibodies for detection of the fusion protein or for your protein?

---

Hi

Actually the fusion protein is the activation domain of GAL4. This constructs are used to do mammalian two hybrid assay (to check whetehr 2 proteins interact).
As the system is not working not even with my positive control, I thought there might be something wrong with the translation of this fusion protein.

The antibody I use is specific for the protein I cloned in not the N-terminal GAL4.

Any idea?

---

hmmmm, a ten kDa difference is pretty big. if your antibodies light up for both bands and the second band is below the one with the correct MW, it's possible that the protein is being degraded. maybe your protein of interest has a domain connected by a flexible linker region that easily falls off. or maybe it's just chock full of protease sites.

another cool Expasy site to check for possible protease cutting sites:
http://us.expasy.org/tools/peptidecutter/

try to see if any of the sites match up to a possible cut site that would leave a 10kDa difference.

---

Hi
I still couldn't solve this problem!
One of the vectors from the mammalian 2 hybrid system , the pBIND (from Promega) which is supposed to encode a N-terminal GAL4 fusion protein, in western I see 2 proteins. This is independent from the gene I clone in there. I cloned to different genes and both gave me 2 proteins, both bands recognixed with an antibody specific to the gene I cloned.

The other vector, pACT, which encodes an N-terminal VP16- fusion protein gives only one protein product, although with a higher molecular weight than predicted.

I sequenced both vectors since the promoter until the stop codon of the gene I cloned and I couldn't find any mistake.

Does anyone have any suestion?

Thanks

---