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
2) T7 promoter - fMet - GS - 6xHis - GSGSGSRRASV ....(the protein of

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

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

Best regards,

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

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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