no expression of my target protein - (Nov/26/2008 )

Hi,
i work with chitinase (cht1) from fungus. i already successfully cloned the gene into expression vector pet22b and trasformed it into expression host, BL21(DE3). After sequencing,everything is in frame.However, after induction with 1mM IPTG and further incubated at 30degree for 2hours, no expresson occured (no band on my SDS-PAGE) when i checked the total cell protein.i alredy tried other range of temperature (20 and 15) but still no positive result.what should i do to solve this problem?plzz help me..

-dinaz-

Hi

Things that u can do:

1) If u want to be sure that the above protein is expressed, u must do Western blotting, before u spend ur time for free. u should

use matching Ab to ur protein tag.

2) Induction time- longer induction time, at list 4h to over night expression.

3)Temp.- try 37C.

4) lower IPTG conc.- try 0.1mM IPTG (4 hours induction)

BYE

thanks amtash.
i will try ur suggestion.any result i will inform later.

Two questions before further suggestions, did you test your inclusionbody of your protein? What lysis buffer did you use?

Generally, the expression test should be considered as bellow, and then try to work out your own protocol:

1. Competent cell test:

If BL21(DE3) doesn't work, try BL21(DE3)pLysS or BL21(DE3)pLysE or Rosetta cells which are derived from B strain.

2. Growth conditions:

What O.D. value did you reach when you added IPTG? Try 0.3, 0.6, 0.8, even 1. Then change different concentration of IPTG (0.5-1mM).

Induction time: try 2,3,4 hours at 37degree. Then 20 or 25 degree overnight (~20hrs)

3. Lysis buffer
What kind of lysis buffer did you use? Try to analyse both soluble part and inclusionbody on gel at the same time