Mammalian expression problem - HEK cells expression (Dec/11/2008 )
Hi,
I have a problem expressing a large protein (more than 500kDa). I use a construct (total ~20Kb) with my gene under CMV promoter (actually, I tried two different vectors). I sequenced the full length of my gene - looks fine. However, it doesn't seem to produce any protein when transfected into HEK cells (I tried Lipofectamine, FuGENE and CaP).
The DNA contains a lot of GC-rich areas, and the protein has stretches of acidic amino acids. Could those factors play a role here?
Will appreciate any suggestions on how to get this protein expressed in HEKs.
A.
-Anetta-
QUOTE (Anetta @ Dec 11 2008, 11:03 AM)
Hi,
I have a problem expressing a large protein (more than 500kDa). I use a construct (total ~20Kb) with my gene under CMV promoter (actually, I tried two different vectors). I sequenced the full length of my gene - looks fine. However, it doesn't seem to produce any protein when transfected into HEK cells (I tried Lipofectamine, FuGENE and CaP).
The DNA contains a lot of GC-rich areas, and the protein has stretches of acidic amino acids. Could those factors play a role here?
Will appreciate any suggestions on how to get this protein expressed in HEKs.
A.
I have a problem expressing a large protein (more than 500kDa). I use a construct (total ~20Kb) with my gene under CMV promoter (actually, I tried two different vectors). I sequenced the full length of my gene - looks fine. However, it doesn't seem to produce any protein when transfected into HEK cells (I tried Lipofectamine, FuGENE and CaP).
The DNA contains a lot of GC-rich areas, and the protein has stretches of acidic amino acids. Could those factors play a role here?
Will appreciate any suggestions on how to get this protein expressed in HEKs.
A.
You may want to try some RT-PCR to see if the message is at least getting made - then you could decide whether it is a problem at the RNA or protein level.
-smu2-
QUOTE (smu2 @ Dec 12 2008, 05:04 AM)
QUOTE (Anetta @ Dec 11 2008, 11:03 AM)
Hi,
I have a problem expressing a large protein (more than 500kDa). I use a construct (total ~20Kb) with my gene under CMV promoter (actually, I tried two different vectors). I sequenced the full length of my gene - looks fine. However, it doesn't seem to produce any protein when transfected into HEK cells (I tried Lipofectamine, FuGENE and CaP).
The DNA contains a lot of GC-rich areas, and the protein has stretches of acidic amino acids. Could those factors play a role here?
Will appreciate any suggestions on how to get this protein expressed in HEKs.
A.
I have a problem expressing a large protein (more than 500kDa). I use a construct (total ~20Kb) with my gene under CMV promoter (actually, I tried two different vectors). I sequenced the full length of my gene - looks fine. However, it doesn't seem to produce any protein when transfected into HEK cells (I tried Lipofectamine, FuGENE and CaP).
The DNA contains a lot of GC-rich areas, and the protein has stretches of acidic amino acids. Could those factors play a role here?
Will appreciate any suggestions on how to get this protein expressed in HEKs.
A.
You may want to try some RT-PCR to see if the message is at least getting made - then you could decide whether it is a problem at the RNA or protein level.
Your protein might be unstable right after its translation, or just due to low expression level since it's quite a large protein. RT-PCR can give you an idea in a short time.
-kingsupercat-
QUOTE (Anetta @ Dec 11 2008, 11:03 AM)
Hi,
I have a problem expressing a large protein (more than 500kDa). I use a construct (total ~20Kb) with my gene under CMV promoter (actually, I tried two different vectors). I sequenced the full length of my gene - looks fine. However, it doesn't seem to produce any protein when transfected into HEK cells (I tried Lipofectamine, FuGENE and CaP).
The DNA contains a lot of GC-rich areas, and the protein has stretches of acidic amino acids. Could those factors play a role here?
Will appreciate any suggestions on how to get this protein expressed in HEKs.
A.
I have a problem expressing a large protein (more than 500kDa). I use a construct (total ~20Kb) with my gene under CMV promoter (actually, I tried two different vectors). I sequenced the full length of my gene - looks fine. However, it doesn't seem to produce any protein when transfected into HEK cells (I tried Lipofectamine, FuGENE and CaP).
The DNA contains a lot of GC-rich areas, and the protein has stretches of acidic amino acids. Could those factors play a role here?
Will appreciate any suggestions on how to get this protein expressed in HEKs.
A.
how do you check expression? try a reporter gene, f.i. in a bicistronic expression vector...
-The Bearer-