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
