4 replies to this topic

[yyzhang]

i have cloned my gene into the pBT-vector,but the problem is that i can not detected the express of the protein on the SDS-PAGE agar.the concentration of IPTG is 50uM.OD is 1.0.what is the problem?help me please.

[phdconsult]

Induction must be tried at 32C/37C and 39C

IPTG can be raised to 75uM

Reduce volume of media per flask ( 40 ml in 250 ml flasks).

Reduce rpm of flask rotation to 10 or 0.

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Cells are under stress when producing a foreign protein and due care must be provided to stabilize the production environment.

[InvisibleSurfer]

Also, try additional induction conditions. Ask yourself, what factors affect protein expression?

[yourself]:

1. Culture volume & growth medium
2. OD of induction
3. length of incubation after induction
4. IPTG concentration
5. Temperature
6. The universe


so the answers to these are:

1. IT'S ALWAYS TRICKY WHEN SCALING UP PROTEIN EXPRESSION, keep that in mind. Try 50ml (or 40ml like phdconsult said), 100ml, 400ml. Also, try LB, 2YT and 5YT

2. Cultures in less rich media (eg LB) need to be induced at lower ODs (try .3 and .5). For 2YT try .5 and 1.0. For 5YT try .5, 1.0 and 1.3. KEEP IN MIND though, small cultures will NOT get to very high ODs, ie you may find that a 50ml 2YT culture will not get any higher than OD 1.5.

3. Take samples after induction at 1h, 2h, 4h, 6h (if possible) as well as after overnight incubation at desired temperature, to determine after how much time you are getting best expression

4. Try 0.1mM to 1mM in steps, like .1, .3, .5, .7, 1.0 (although in smaller than 500-1000ml cultures, IPTG concentrations higher than .3 do not make any difference)

5. In regards to temperature, like phdconsult said, try room temperature (22-25'C, or bellow 30'C), 37'C and higher. Expression in low temperatures also increase the chances of your protein remaining soluble.

6. Not much you can do about that

Scalling up protein expression is a b-i-t-c-h, I've had huge problems, don't be afraid to try different things and go for different growth conditions

To phdconsult:

What's so special about induction at 39'C ?

Thanks

[phdconsult]

To phdconsult:

What's so special about induction at 39'C ?

Thanks
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Invisiblesurfer:

37C is the ambient temperature. 39C is stress, but not 'shocking stress'. At that temperature, cells make a determination to produce non-housekeeping proteins including proteases, HSPs etc and our cloned protein is somehow included in the list. At 42C, the cells tackle it as a known heat shock challenge and our foreign protein goes down on the list of 'to be expressed' products.

[InvisibleSurfer]

phdconsult, thank you for the prompt reply.