Expressing an SSB protein

Marco Candeias marcomarques at netzero.pt
Tue May 8 14:35:18 EST 2001


Subclone ORF.
pET system vectors usually work for controled expression with IPTG. You
might need to use pLysS or E as well.

Mike Powell <mikepowell at mindspring.com> wrote in message
news:9d7rb7$6e$1 at slb1.atl.mindspring.net...
> Martin,
>
>   Personally, I would subclone your ORF into a good
> expression system.  I have expressed phage ssDNA
> binding proteins in T7 systems with luck.
>
>   Look again at the literature.  There are plenty of
> papers describing the cloning and expression of
> ssDNA binding proteins.  Attached is one example
> that uses His tags.  But you could also use ssDNA
> Cellulose affinity columns if you don't want the His
> tag.
>
>   Good luck!
>
> -Mike-
>
> Protein Expr Purif 1999 Jun;16(1):96-102
>
>
>                        Cloning, overexpression, and purification of the
> recombinant His-tagged SSB protein of Escherichia coli
>                        and use in polymerase chain reaction amplification.
>
>                        Dabrowski S, Kur J.
>
>                        Department of Microbiology, Technical University of
> Gdansk, ul. Narutowicza 11/12, Gdansk, 80-952, Poland.
>
>                        Polymerase chain reaction (PCR)-derived DNA
fragment
> containing the complete structural gene for SSB protein of the Escherichia
>                        coli was cloned into an expression vector. The
clones
> expressing His-tagged SSB protein were selected. The cloned DNA fragments
>                        were verified to be authentic by sequencing several
> clones. The recombinant SSB protein (His-tagged SSB) contained a
> polyhistidine
>                        tag at the N-terminus (38 additional amino acids)
> that allowed single-step isolation by Ni2+ affinity chromatography. We
found
> that
>                        recombinant plasmids are unstable and give a low
> level of expression in E. coli BL21(DE3) strain. However, the plasmids
were
> stable
>                        in E. coli BL21(DE3) containing the pLysS plasmid,
> which suppresses expression prior to induction, and His-tagged proteins
were
>                        highly expressed upon IPTG addition. The SSB
protein
> was purified by metal-affinity chromatography on Ni2+-TED-Sepharose
>                        columns. The enzyme was characterized by
fluorescence
> titration experiments for single-stranded DNA binding activity. We have
>                        applied the use of His-tagged SSB protein to
increase
> amplification efficiency with a number of diverse templates. The use of
SSB
>                        protein may prove to be generally applicable in
> improving PCR efficiency. Copyright 1999 Academic Press.
>
>                        PMID: 10336866 [PubMed - indexed for MEDLINE]
> "martin murray" <martym at _remove_altavista.net> wrote in message
> news:D4C35DEF26CAD36D.0BF7015486D4EC2F.B4C2B5EE0D38C860 at lp.airnews.net...
> > We have identified a gene in an unculturable bacterium which appears
> > to code for a single-stranded DNA binding protein. It is part of a
> > larger plasmid fragment and has been cloned into pUC18. We are
> > interested in possibly expressing this gene, purifying the protein,
> > and determining whether it will bind SS DNA in vitro.
> > Not having done this before, or anything like it, we have some
> > questions:
> >
> > Which of the many available expression kits would be best for this
> > experiment?
> >
> > Is it necessary to cut out the relevant ~400 bp ORF and reclone that
> > into the expression vector,
> > or can we use the pUC18 construct we already have?
> >
> > What is involved in the protein purification process?
> >
> > What conditions are required for the actual DNA-binding step?
> >
> > I have looked for answers to these questions on the internet but
> > haven't had much luck, and there
> > is no one at this facility to ask. Thanks very much for any advice you
> > can give.
> >
> > MM
>
>





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