protein expression in BL21 strain
khuang at BIOC.RICE.EDU
Fri Dec 4 10:12:04 EST 1998
try to use ER256 (NEB) or Strategene one (sorry forgot the name), both of
are endonuclease mutant.
On 4 Dec 1998, Alexandre Mooser wrote:
> I cloned 2 fragments (hydrophobic C-terminus of proteins) into the
> vector pTFT74, which is under control of the T7 promoter. The constructs
> are expressed in BL21(DE3), which is a particular strain of E.coli used
> for protein production in inclusion bodies. I also transformed XL-I-Blue
> cells to sequence my constructs.
> When I isolate the DNA from XL-I-Blue, everything looks ok (bands at
> right height on DNA gel). Sequencing is ok (right frame). I transformed
> then BL21(DE3) cells with this same DNA (same clone), and isolated the
> DNA to check if everything is still ok.... and problems appeared: the
> constructs seem to be degraded (bands 30% shorter on agarose gel). Of
> course, my protein is not expressed (checked on SDS-PAGE) !!.
> Very frustrating ! I repeated the whole many times, and the same happens
> each time. Can anybody help me ??
> Did anybody already work with BL21(DE3) cells ??
> Any suggestion welcome
> Alexandre Mooser
> Institute of Biochemistry
> University of Zurich
> amooser at biocfebs.unizh.ch
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