Tnt system-in vitro translation

Chris Noren noren at neb.com
Mon Aug 15 14:30:47 EST 1994


In article <32e4d8$iml at network.ucsd.edu>, T. S. Pillay <tpillay at ucsd.edu>
wrote:

> I would like to use the Tnt system or any other in vitro translation
> system to make protein from a plasmid which has both the T7 promoter and
> the Sp6 promoter. Which is better?
> 

TnT system from Promega works great!! There's no reason why SP6 or T7 would
be better except that the kit comes with T7 polymerase.

> Has anyone used the tRNA ascend system to make biotinylated proteins in
> vitro?

Long before the availablity of Promega's tRNAscend system (or the
Boehringer equivalent), we set this system up in my own lab. We encountered
three problems that neither Promega nor B-M address:
1) As more biocytin-tRNA is added, more lysines in the protein being
synthesized in vitro are replaced with biocytin (biotinylated lysine),
which is significantly larger than lysine. This results in increased
molecular weight of your protein, depending on the desired signal
intensity.
2) The "label" is prepared by first enzymatically aminoacylating tRNALys
with lysine, and then chemically modifying the e-amino group of the lysine
with biotin. Trying to specifically derivatize an amino group with pKa 11.8
while leaving the a-amine (pKa = 9.18) alone is a chemist's nightmare,
especially when the molecule being derivatized has a molecular weight in
excess of 26 kDa. It is thus very difficult to characterize the products of
the modification reaction, and we observed significant batch-to-batch
variability in our hands.
3) The detection system will pick up any endogenous biotinylated proteins
in the cell extract being used for translation. For an E.coli-based system,
the biotinylated subunit of acetyl-CoA carboxylase (22 kDa) gives a very
intense band using streptavidin-based detection systems, right in the
middle of where our target protein was supposed to be.

I'll stick with S-35. :-)

-- 
Chris Noren

We Await Silent Trystero's Empire



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