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pMal-c2, modification of

ChenHA hzhen at freeuk.com
Fri Jan 28 20:49:39 EST 2000


Wolfgang Schechinger wrote:

> Hi all,
>
> I would like to express a His-tagged protein in E. coli. The only
> suitable vector I have at the moment, is pMal-c2.
> There is a unique restriction site (NdeI) right
> between the Ptac promotor and the start of malE translation.
> Thus I would cut out the whole maltose binding protein (and get rid
> of it since I don't need it anyway, because I have a His-tag) and
> insert my construct between this NdeI site and a the polylinker.
>
> Despite all this sounds quite reasonable to me, does anybody have
> any caveats, drawbacks or warnings to mention?

I haven't use pMal-c2 before, so I'm only guessing as to what you are
doing.  I would presume that you are in effect expressing the protein
directly without the presence of a fusion partner, using the ATG of the
NdeI restriction site as the start codon for your protein.  As long as
the start codon ATG is at a suitable distance from the RBS, the
construction should be OK.

However, you should be aware that the presence of a fusion partner is not
just only for purification.  The presence of a fusion partner helps with
solubility, protein expression, stability, etc.  One thing that you
should bear in mind is that without the fusion partner, your protein may
express poorly due to inefficient translation initiation.  This very much
depends on the initial DNA sequence of your gene.  A high GC content at
the translation initiation region of your coding sequence is generally
bad.

>
> All input is welcome!
>
> Wolfgang
>
> For those who don't know pMal-c2:
> pMal-c2 is a glucose inducible vector for procaryotic protein
> expression. When used normally, one has a fusion protein between
> maltose binding protein (malE) and the protein on interest; the
> bacterial lysate is purified by affinity purification using a amylose
> (maltose-like polymer) resin. Then the fusion protein is cleaved off
> proteolytically with factor Xa.
>
> Dr. Wolfgang Schechinger, Pathobiochemistry Dept.
> University of Tuebingen, Germany
> email: wolfgang.schechinger at med.uni-tuebingen.de
> wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
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