Q. About Protein Expression !!!

Randy Willis willis at gandalf.psf.sickkids.on.ca
Tue Mar 11 14:15:28 EST 1997


ELLA CHAN wrote:
>   Could somebody kindly tell me at which OD600 will be the optimum
>   condition for the induction of protein expression by using IPTG
>   in bacterial strain ?

Ella,

If only it were that simple.  The optimal point of induction is going to
be dependent upon a large number of factors, not the least of which is
your protein.  My advice would be to perform an induction trial
experiment whereby you start with a large culture (ie 1l) and you can
remove and induce aliquots of the culture at different OD600 readings
(ie 0.3, 0.6, 0.9, 1.2, etc).  As well, once the cultures are inducing,
you will want to take small samples of the growths at different times to
see what is the optimal growth time post-induction for your system.  You
can easily boil pellets from 0.5ml samples by adding SDS-loading buffer
to the pellet.  I've always tried to add sample buffer in a ratio with
the OD such that volume sample buffer (ml) = OD600/20 (for a 0.5ml
growth sample).   This tends to give even gel loading between lanes and
gives sufficient protein concentration to prevent smearing in gels. 
Boil for about 5 minutes, vortex for 30 seconds and microfuge for 2
minutes.  If the sample is slimy when you try to pipette it, this is
usually due to nucleic acids and can be reduced by vortexing longer and
centrifuging again.

Other factors to be considered when looking for optimal expression, a
bacterial strain choice, temperature of growth and media recipes. 
Different bug strains cope with different protein expressions, well,
differently.  Also, some proteins may be lethal to or degraded by
different cell lines and this may be limited by growth and expression at
lower temperatures (ie 30C, 25C, 20C...I've even seen growth at 4C). 
Also, mid-log, late-log and stationary phases occur at different OD's in
different media such that OD600=1.2 is late in minimal (M9) but will be
mid in rich media (LB) or early in very rich media.

If inducing in a medium which allows growth into very high OD's, watch
for pH changes as media may acidify after several hours and this will
interfere with growth.  I like to buffer with 1M potassium phosphate,
tribasic.  Also, you may want to add extra glucose as the growth reaches
a high OD, but only if you want the growth to continue and you think
that induction is still proceeding.  If you do go for long inductions
(including perhaps overnight growth), watch for any effects that this
may have on protein solubility.  I have seen a few examples where a
protein which was soluble a few hours post-induction, is exceedingly
insoluble after overnight induction.  Temperature of induction can also
be a factor in this.

As you can see, there are a lot of things to consider with a biological
system and I can promise you that there are few hard and fast rules.

Good luck,

Randall C Willis, Researcher
Biochemistry, Hosp for Sick Children
3522-555 University Ave
Toronto, ON
M5G 1X8  CANADA

416-813-5933 (ph)  -5022 (fax)

willis at gandalf.psf.sickkids.on.ca



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