Loss of expression, help!

Troels Wind wind at biobase.dk
Tue Nov 7 08:02:23 EST 2000


Dear all,

I have a rather annoying problem with my coli expression system that I cant
explain, why I hope some of you may have an idea.

I have my protein in two different, but similar, vectors with the following
compositions of the promoter/initiation region:

1) T7 promoter - fMet - GSMGSCGS - 6xHis - IEGRV ....(the protein of
interest)
2) T7 promoter - fMet - GS - 6xHis - GSGSGSRRASV ....(the protein of
interest)

The vector is basically a pET-vector and is the same for the two constructs.
Number 1 has a free Cys (no Cys in my protein), hexa-His and a fXa
proteinase-site (IEGR).
Number 2 has hexa-His, a Gly-Ser linker and a HMK-recognition site for
Ser-phosphorylation (RRASV).

The tricky thing is, that construct number 1 works perfect in BL21(DE3) as
well as BL21(DE3)pLysS (~10 mg protein per liter culture) whereas number 2
is lousy (~0.1 mg per liter).

It is not a question of codon-choice; all codons are selected for optimal
coli-expression.

Does anyone know what is going on? Any input is much appreciated!

Best regards,
Troels

Troels Wind, Ph.D.
Laboratory of Cellular Protein Science
Department of Molecular and Structural Biology
University of Aarhus
Gustav Wieds Vej 10C
8000 Aarhus C
Denmark

Phone: (+45) 8942 5079
Fax: (+45) 8612 3178
Mobile: (+45) 2856 9767

"Only two things are infinite, the universe and
human stupidity, and I'm not sure about the former."
                                                   -Albert Einstein


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