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
