fusion protein construction

Cathal Garvey via methods%40net.bio.net (by cathalgarvey from gmail.com)
Sat Oct 3 18:01:53 EST 2009

Hi Azam,
If you just want to have both genes active in the transformed cells, then it
is not necessary to fuse them. Doing so might inactivate the function of one
or both.

If, on the other hand, you aim for them to perform a function together, then
fusion may indeed be a good way to do it. Fused proteins have been observed
to provide far greater activity than the separate proteins might if
co-expressed, assuming they work upon the same substrate. The linker region
should be at least 8-10 amino acids long, I think, assuming the C-terminal
end of the first protein and the N-terminal end of the second protein are
both exposed on the outer surface of the proteins (if in doubt, they
probably are). Try to pick inert amino acids that don't force structural
changes due to their shape. A pair of simple uncharged amino acids repeated
4-5 times should do, I think.

I hope this helped! Also, don't take it as authoritative. I've only read a
few papers on the matter.

2009/10/3 Azam Rahimpour <rahimpour_a from yahoo.com>

> Hi
> I am going to construct a gusion protein between an enzyme (alkaline
> phosphatase) and a neomycine resitance gene for expression in mammalian
> cells, I study in some articles that in some cases a linker sequence is
> added between two proteins, please let me know if:
> 1. when create a fusion protein what is the risk that individual proteins
> loss their activity
> 2. is adding a linker necessary and what kind of linker works best
> regards
> azam
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