C-terminal GST fusion problem in mammalian cell - (Feb/04/2013 )

Hi, guys. I have some problem with protein expression in mammalian cells. Any help will be appreciated!!

1. I am expressing a protein which has a GST at the C terminal (must at C terminal) in HeLa cells. Do I need to add ATG before GST gene?
2. Is there any tag that is better (at expression level) at C terminal?
3. Some book says if there is a restriction site (especially BamH I) near ATG start codon, the translation is pool in insect cells. Is that ture in mammalian cells?
4. I use pcDNA3.1. The mannual recommend adding Kezak sequence (G/ANNATGG). Mine is AGCATGC (the last one has to be C). Will that affect translation a lot?


I cannot see a band when doing western blot in HeLa cells... My transfection efficiency is ~50%. I can see anti-GFP band. My GST antibody definitely works. Please help me...

Or anyone has some tricks when express protein in mammalian cells? Thanks a lot!

snowchild

You shouldn't need the ATG at the start of the GST, but it shouldn't matter.

GST is relatively large, if you are not going to be purifying the protein, then FLAG or V5 are good alternatives. However, GST is very soluble and can stop proteins entering the insoluble fraction.

I don't know about the RE site thing, never heard that before.

The kozak sequence can help, but is not absolutely necessary. The C after the ATG may lower the expression, but this is not necessarily the case.

If you can't see the band - what conditions have you tried for lysis? Do you have a GST +ve control lysate to ensure that the antibody is working? How much protein are you loading? What promoter is on the plasmid?

Thank you so much, bob1!

I will purify it. My peptide is only 1-2kD, and that's why I use GST.

I use 500ul M-Per reagent to lysis one 10-cm plate of cells. I used GST purified from E.coli as a + control. I loaded 1/100, 1/50. 1/25 on the 0.75mm gel. pcDNA5 I used has a CMV promotor. I can see GFP (pcDNA3, only lack a flp in site compare to pcDNA5) band when use the exact same condition.

I also used SDS-PAGE loading buffer to lysis cell. I will see the result soon.

I am trying HEK293T cells now. The plasmid I use can replicate in HEK293T cells.

That should work fine. I think you will find that most plasmids won't be replicated in 293T cells unless they are the EBNA strain of 293.

Thank you, bob1.

I used GST amplified from pGEX (for E.coli expression). Someone told me I should optimize the codon for human. Will that affect a lot?

I still cannot see the western blot result since my reagent hasn't come yet.

Codon optimization can help, but in my experience it usually doesn't matter all that much. I think that optimization for bacteria is more important than for mammalian cell expression.

Thank you.

I decided to make a new construct with other tag, like GFP and His. Forget about C-terminal GST...