kDNA minicircles - forwarded

[Biotério de Animais Genéticamente Manipulados.]

Try to use protease inhibitors like pepstatin and leupeptin in the
extraction buffer, as some proteins are quickly destroyed by cytoplasmic
proteases. Use urea 6M in the sample buffer of Laemli systems of SDS-PAGE,
in order to solubilize the proteins. Change vectors and expression systems,
like protease deficient S.cerevisae 

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De: Janice Taverne <J.Taverne at ucl.ac.uk>
Para: Multiple recipients of list <leish-l at bdt.org.br>
Assunto: kDNA minicircles - forwarded
Data: Terça-feira, 24 de Março de 1998 08:38

>From: root at cscdri.ren.nic.in
>>From: Superuser <root at cscdri.ren.nic.in>
>To: j.taverne at ucl.ac.uk
>Date: Tue, 24 Mar 98 8:51:15 PST
>
>        Since I cannot access the parasitology biotech forum
>directly from my end, that's why I am writing to you to
>kindly float my following query for an answer, to parasitology
>colleagues.
>        During the course of my DNA sequencing of kDNA minicircles
>of an isolate of Leishmania, I have come across an open reading
>frame. It is of 93 a.a. and codes for an extremely hydrophobic
>putative protein of 10 kD. An ORF in the variable region of
>minicircles is highly surprising to me as I thought that they do
>not code for proteins. But by northern blot analysis I could even
>detect a transcript! This then prompted me to express the ORF in
>E. coli. I got the expression library with the insert in the right
>orientation etc but I cannot see the protein on SDS-PAGE. Could
>it be that the protein is not finally translated or is its extreme
>hydrophobicity giving a problem? Looking forward eagerly to some
>suggestions.
>        Neeloo Singh (root at cscdri.ren.nic.in)
>