Overexpressing bacterial protein in mammalian cells - which vector to use? - overexpression in mammalian cells (Dec/07/2006 )

Hello all experts,

I am trying to overexpress bacterial proteins in mammalian cells.

I need the following information:

1) which is the best cell culture system to use for protein overexpression?

2) which is the mammalian vector to use for cloning my gene of interest (coding for bacterial protein)??

3) which is the best promoter to drive expression in mammalian cells??

4) any other considerations to keep in mind??

I look forward to all your expert advise.

thanks.

1) best is Hela cells in suspension if you want to obtain big amounts of the protein. Adherent is better if you want to study effects. So cell line is HeLa S3 which hold the double capacity

2) i would recommend pCMVflag vector or pFHM (one CMV promoter Flag HA and myc tags)

3) well CMV promoter is the most used.

4) inductibility of the expression may be relevant if your protein induce cytotoxicity

Thank you very much.

I hear that 293F cells grow very well in suspension and are ideal for overexpression in mammalian cells. Is this true?j

Also, how do Elongation Factor 1-alpha and SV40 promoters compare to CMV??

Thanks again for your great suggestions.

well pubmed should hepl you about these promoters.
I know that my puromycine resistance is due to SV40 promoter.
I don't hav insights about EiF1 although i've heared about it.

I have HelaS3 cells but 293F (F for freestyle) are ok too

How is pCDNA vector?
Just because we have it on our department floor.

If CMV promoter is good for overexpression, then I need not clone my gene of interest into pEF4 vector with the EF1 promoter.

We also have 203F cells on our department floor, so I will be use this and use pCDNA for transient transfection and overexpression in suspension cultures.

Pl. let me know.
Thanks.

How crucial is the Kozak sequence?

should I incorporate this sequence in my upstream primer for cloning my gene of interest??

thanks.

well an easy google search give this for pCDNA. I assume you have sthg similar. Yes CMVis good for overexpression and i use it currently.

The technical manual for pCDNA3.1 says that "pCDNA is a "non-fusion vector" and your insert must contain the kozak sequence and the ATG start codon for proper initiation of translation."

Were you refering to something different when you said that kozak is not needed in amplification?!

thanks.

Yes, it is mentioned!! On page 2 of the Methods section under the sub-heading "Cloning considerations" (page 8 of the pdf file).
I appreciate your comments.
thanks.

well i've used the CMV promoter for expression of a protein and didn't add the kozak sequence... although the protein is expressed.
in link i posted the manual doesn't speak about a kozak sequence?.

i've deleted the post in which i mention that kozak sequence is not needed as i don't want to mislead other users and esp for you, please accept to forgive me for that big mistake.

I've searched in my constructs sequences as finally my exp let me do that properly and here is the result :
iin the sequence below you'll please find
pCMV immediate early promoter used in my pFHM vector (1-589)
the KOZAK sequence used (please forgive me ) 687 to 698
the flag epitope starting at 699 till 722
as the kozak sequence was inserted in the original vector, i didn't had to add it in my constructs. That was why i supposed that it was not necessary.

so the kozak sequence is needed


Here may you please find the sequence of my construct :
1 GTTGACATTG ATTATTGACT AGTTATTAAT AGTAATCAAT TACGGGGTCA
51 TTAGTTCATA GCCCATATAT GGAGTTCCGC GTTACATAAC TTACGGTAAA
101 TGGCCCGCCT GGCTGACCGC CCAACGACCC CCGCCCATTG ACGTCAATAA
151 TGACGTATGT TCCCATAGTA ACGCCAATAG GGACTTTCCA TTGACGTCAA
201 TGGGTGGACT ATTTACGGTA AACTGCCCAC TTGGCAGTAC ATCAAGTGTA
251 TCATATGCCA AGTACGCCCC CTATTGACGT CAATGACGGT AAATGGCCCG
301 CCTGGCATTA TGCCCAGTAC ATGACCTTAT GGGACTTTCC TACTTGGCAG
351 TACATCTACG TATTAGTCAT CGCTATTACC ATGGTGATGC GGTTTTGGCA
401 GTACATCAAT GGGCGTGGAT AGCGGTTTGA CTCACGGGGA TTTCCAAGTC
451 TCCACCCCAT TGACGTCAAT GGGAGTTTGT TTTGGCACCA AAATCAACGG
501 GACTTTCCAA AATGTCGTAA CAACTCCGCC CCATTGACGC AAATGGGCGG
551 TAGGCGTGTA CGGTGGGAGG TCTATATAAG CAGAGCTCTC TGGCTAACTA
601 GAGAACCCAC TGCTTACTGG CTTATCGAAA TTAATACGAC TCACTATAGG
651 GAGACCCAAG CTTGGTACCG AGCTCGGATC GATATCGCCG CCGCCATGGA
701 CTACAAGGAC GACGATGACA AGTACCCTTA