Expressing an SSB protein
marcomarques at netzero.pt
Tue May 8 14:35:18 EST 2001
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...
> 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
> Good luck!
> 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
> containing the complete structural gene for SSB protein of the Escherichia
> coli was cloned into an expression vector. The
> 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
> tag at the N-terminus (38 additional amino acids)
> that allowed single-step isolation by Ni2+ affinity chromatography. We
> recombinant plasmids are unstable and give a low
> level of expression in E. coli BL21(DE3) strain. However, the plasmids
> in E. coli BL21(DE3) containing the pLysS plasmid,
> which suppresses expression prior to induction, and His-tagged proteins
> highly expressed upon IPTG addition. The SSB
> was purified by metal-affinity chromatography on Ni2+-TED-Sepharose
> columns. The enzyme was characterized by
> titration experiments for single-stranded DNA binding activity. We have
> applied the use of His-tagged SSB protein to
> amplification efficiency with a number of diverse templates. The use of
> 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
More information about the Methods