400 ml is a lot in a 1 liter flask. Use 1/4 to 1/5 of the flask volume.

may be the BL21 codon plus cells which you are using are not the fresh one. try expression with fresh cells may be this solve your problem.

Is the plasmid constitutive or is the expression induced? it could be the concentration of the inducer( or some other nutrient) is off

Did you make a startculture for the 400 ml culture? I usually add 0.5 ml of a startculture (with glucose, OD 0.5) to 100 ml autoinduction medium.

Acetate inhibition could be your problem at the altered scale.....

400 ml is a lot in a 1 liter flask. Use 1/4 to 1/5 of the flask volume.

Go for a fresh transformation and inoculate the culture. Do not exceed your culture volume beyond 200 ml in 1ltr flask.

I our lab we routinely use this strain of E. coli for over expression and do not notice any problems with expression. We also cultures volumes up to 500ml. We use a fresh overnight culture (5mL) and use this as the inoculum into 250 ml. We use 25 ug/ml Kanamycin and agitate as 150 rpm. we use 0.2mM IPTG as the inducer.

It is strange that is expresses in one and not the other, which is why I have just given you our protocol. What temperature are you epxressing at? Sometimes you can enhance protein expression by reducing the temperature to as low as 15oC. That has worked for us a few times.

Hope this helps

Ian

Its but obvious that you are using too much volume of culture in 1 lit flask.

A most use up to 200-250 ml of culture for the 1 lit flask.

It will improve your experiment results.

BOL

Does your BL21 contain tRNA on plasmid? The plasmid (normally chloramphenicol resistant) is used for optimizing codon usage of your gene in E.coli, and may be easier to be lost in large scale culture than in small scale culture due to the longer growth time.

As several people already pointed out, 400 ml cultures should be used in 2L flasks (1/5 volume is a good rule of thumb), or insufficient aeration will alter the glucose/lactose metabolism - which I assume you use for your induction.

In addition, it occurred to me that small and large cultures are commonly grown in different incubator-shakers, and if that is the case, it is worth checking the exact temperature in each. Even 2-3oC could make a difference to some proteins. A sensible control would be to take a small aliquot of the large culture into a small vial and shake them in the same incubator.

Good luck, and please post when you find out the answer!

As already mentioned aeration could make the difference, but we also observed that differences in the preculture can affec efficient Proteinexpression. Try to keep the cells exponential when starting the main culture.

Good luck

Definitely not more than 200 mL culture in a 1 Litre conical flask. use a fresh colony from an overnight incubated plate, make a 2 mL starter culture from a single colony, don't grow it more than slightly turbid (OD less than 0.4) and inoculate the bigger culture. Make no compromises...if you did a successful small scale experiment, and a few days later you scale up but use the same petridish (now several days old) then this is asking for trouble. Always re-transorm your construct, get a single colony and start from that, never do overnight precultures either...