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Slimy recombinant e.coli pellet

P J via methods%40net.bio.net (by pow.joshi from gmail.com)
Thu Nov 16 16:00:54 EST 2017


If the sliminess is due to the DNA released from lysed cells, you have the
following options-

1. use a low-tech method of passing the cell suspension through a large
gauge like say 18G needle a few times followed by a small gauge needle a
few time to shear the DNA, followed by a quick centrifugation step.
2. Use a sonicator to shear the DNA.

Personally, I've used method 1 for eukaryotic step successfully. I'm
guessing it would work just fine for a bacterial suspension as well. This
is also assuming your inclusion bodies won't sediment under quick, regular
centrifugation step.

DK, Wo, other experts, feel free to weigh in.

Pow

On 16 November 2017 at 02:57, Megha Goyal <mgbiotec from gmail.com> wrote:

> dear DK sir,
>
> yes we also assume the same. so any suggestion how can we handle it. Our
> protein is in form of inclusion bodies and we get our protein but slimy
> cell mass makes it difficult to handle the cell paste. Also our induction
> is with IPTG for only 3 hours. so can anyone suggest how do we prevent the
> cell lysis if its occurring as we maintain DO, temperature etc during the
> process and we do feeding with glucose at intervals during the induction
> phase.
>
> On Fri, Nov 10, 2017 at 5:51 AM, DK <dk from no.email.thankstospam.net> wrote:
>
> > In article <mailman.110.1510233337.15473.methods from net.bio.net>, Megha
> > Goyal <mgbiotec from gmail.com> wrote:
> > >Dear All,
> > >
> > >We are doing fermentation for recombinant protein using E.coli culture.
> > >Earlier we used to obtain compact cell pellet on harvesting (3 Hrs after
> > >IPTG Induction). But recently we observed that the cell pellet harvested
> > >are of slimy consistency and they do not homogenise in the washing
> buffer
> > >(tris-nacl-edta). We observe thread like consistency and no matter of
> > >stirring it do we get a homogeneous suspension.
> > >
> > >Can someone guide me what is the reason for such phenomenon.
> >
> > Partial cell lysis. "Slime" = chromosomal DNA.
> >
> > DK
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