On 4 Dec 2000, Phil Harrison wrote:
> Hello all,
>> I have some samples E. coli that have been transformed with a plant
> enzyme. I want to extract the enzyme from a liquid culture of the E. coli
> using mild conditions, rather than harsh denaturants. The only methods
> that come readily to mind, given my equipment and plant science background,
> are freeze-thaw cycles and sonication. I would appreciate help with the
> following questions:
What a chance coincidence... I never visit the
bionet.molbio.proteins newsgroup and I happen
to be transforming some enzymes in E coli right
now, and extracting proteins for enzyme assays.
I wash the cells in PBS several times. I think
the buffer could be pretty much anything. I
need to remove "junk" from the LB medium
before lysing my cells. I spin them down
several times to clean them up.
I then put them into an intracellular (low NaCl,
high KCl) "inverted" buffer which I suspect will
lyse bacterial cells, just as it does
most mammalian cells. I then freeze them
at -80, thaw, do a brief sonication, several
pulses, spin in an Eppendorf centrifuge at
14000 RPM for 20 minutes, collect the
supernatant and I am in business. This extract
displays a very high NADH oxidation activity,
which I suspect is due to PFK upon addition of
fructose-6-phosphate. I am transforming with
mammalian PFK mutants, using added NADH to
monitor PFK activity. I am still learning as
I go and welcome suggestions, but I think this
protocol is ok.
Some warnings....
Yali Li shows in J Biol Chem, 269(8)5781-87. 1994
that some proteins will be trapped within the cell
pellet and your protein may end up dispersed between
the pellet and the supernatant. Further it is
mentioned that you can improve your yield by
growing at 30 degrees rather than 37 and using
some additives like sorbitol.
Hope I wrote something helpful and soon enough...
Cheers,
Dominic
North 59 37' 30"
East 17 48' 10"
