Stable expression of very large ( >250 kDa ) proteins in human cells

[Aawara Chowdhury]

In <mailman.1462.1181706938.5139.methods from net.bio.net>,
 Ozan Aygun <metugenetics from yahoo.com> wrote:

> Dear all,
>
> However, some of the proteins that I am interested in
> are above 250 and 300 kDa. I have cloned and
> transiently expressed these large clones and observed
> their expression by WB. Furthermore, when I have
> proceeded for the selections in 1 ug/ml puromycin in
> 293 cells, I have obtained several clones as usual. 
> Unfortunately, when I assay these puro resistant
> clones for the expression they seem to be either not
> expressing or expressing a shorter form of the
> protein.
>
> Since in IRES bicistronic system theoretically a
> resistant clone should express the protein in frame, I
> have been really puzzled with this unexpected
> situtation.

Not surprising. The most likely possibility is that when your construct 
has randomly integrated into the chromosomal DNA, the break-point in your 
plasmid is within your gene.

If the insertion point on chromosomal DNA is within a transcribed region 
(or close to a promoter), the cells will be puro-resistant, and express
a shorter version of your protein (if inserted in-frame with an upstream
ATG), or not express your protein at all (more likely).

> Here are my questions regarding this confusing issue:
>
> 1. What can I do best in this situation to get an
> expressing clone? I.e: do you think increasing the
> puro concentration might help? or should I select,
> propagate and screen more and more colonies? or Should
> I give up this and try a completely different
> approach?

You have several choices.  Here are two:

1) Express your protein using an AAV vector.  These vectors can have
inserts as large as 10 - 12 kb.  They are integrated into one (or two)
chromosomal locations via the AAV ITRs.  You should be able to express
a 3000 a.a. protein using an AAV vector as long as the protein is 
expressed from a cDNA.

2) Use an oriP plasmid.  These are stable, episomal vectors that are
maintained at 1 - 50 copies per cell (depending on cell-type and 
initial transfection efficiency).  OriP plasmid can have inserts of
100 - 200 kb.  Invitrogen used to sell them.  If they no longer do,
contact Bill Sugden at the University of Wisconsin for a suitable oriP 
plasmid.  Or Ashok Aiyar at Louisiana State University.

AC
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