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pDEST42 induction fail

protein expression

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2 replies to this topic

#1 @bhijit

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Posted 23 November 2011 - 04:44 AM

Hi friends,


I am trying to induce 4 proteins of size 15-40 kDa using plasmid pDEST42 and expression host - BL21[DE3] plysS

Procedure:
from 5 ml overnight culture I use 150 to add in 15 ml cultures -
When the OD 600 nm is 0.6 I induce with 0.2, 0.5 and 1 mM IPTG - expression is checked at 4 hr and overnight.

For protien of size 15kDa previous publications have used pDEST with BL21SI cells and got good induction

For protein of size 30kDa previous publications have used pRSET and BL21[DE3]pLysS cells and got good induction


BUT I HAVE NOT GOT ANY INDUCTION IN MY INDUCTION TRAILS :(


i was thinking i dont have same combination of plasmid and expression host - do you think this can affect the induction of protein

Additional information: the colonies on plates look fine on overnight growth. The IPTG is made newly.
Only one mistake i did is the chloremphenicol stock - it is suggested 100% ethanol to be used - but somehow i have made the stock in 50% ethanol - i dont know if this can affect the result

Eagerly waiting for responses

#2 @bhijit

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Posted 30 November 2011 - 05:48 PM

OK I looked for the plasmid sequence - it has lacI repressor -

I guess the genome lacI repressor + plasmid lacI repressor + plys S plasmid is a too much repression

I use highest IPTG 1mM - i was wondering if increasing the IPTG will make any difference

How much maximum IPTG can we use in the expression trail ??

#3 @bhijit

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Posted 30 November 2011 - 05:52 PM

Also I have one doubt about sonication - For 5 ml cell pellet I use 500 ul PBS and sonicate with 50% amplitude

10 sec each pulse - total 5 pulses -- usually the pellet should be very few proteins ? right ? I can see almost similar pattern to soluble fraction ?

Am I doing the sonication correctly ???





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