mammalian expression vector

From: Jerry McCully
Date: 14 March 2012 02:36

Dear ALL;

      As an alternative strategy to avoid endotoxin, I plan to express the protein in mammalian cells.

      As suggested by others, the typical vector is pcDNA3.1(+).  Does anyone have comments on this vector or recommend some other powerful vectors?

     I am new to mammalian expression. I designed a Kozak sequence followed by a BSA signal peptide in order to clone the target into pcDNA3.1(+).

      Is it right?  Tentative Kozak sequence:  GAGCTCGGATCCGCCACCATGAAGTGGGTAACCTTTCTCCTCCTCCTCTTCATCTCCGGTTCTGCCTTTTCT

       Thanks a lot,

Jerry

----------
From: A. Radu Aricescu
Hi Jerry,

You may find that pcDNA3.1 won't give you the protein yields needed for crystallization. Have a look at PMID: 17001101 for an alternative. Your Kozak sequence looks good,

radu

------------------------------------------
A. Radu Aricescu, PhD