5 replies to this topic

[Pangea]

Hi,

how can İ best get in E. coli or Mammalian cells an protein expression yield of more than 1g/l?

Any kind modifications?

Cheers

[Adrian K]

How do you optimize your expression in the first place?

[Pangea]

It will be a pET vectorsystem for E coli. One can play with media and ZnCl2, Glucose and VPA. But is that enought for very high yield production.?

[Adrian K]

I tried media replacement before induction. It is relatively yields higher expressed product than just merely induction with old media with / without modification.

[Pangea]

So you mean replacement by centrifuging the cells and renewing the media. Can you give me more details like an protocol?

[Adrian K]

Yes.

Based on my memory, First I grow my E. coli till it reaches a certain OD600 value I want (I usually grow it to 0.5, some protein I still can express it very well at 1.0), centrifuge 5 minutes at 4'C with 8000g to pellet the bacteria cells,
I will then replace the media with final concentration of 0.5mM IPTG to induce the protein expression. My cells growing usually took 3-4 hours (37C), and protein expression usually took another 4 hours (~30C).

p/s: When I am Growing the cells I do include antibiotic. But when I do expression, I excluded it totally. Haven't really verified by myself, but I believe production of lactamases will reduces the availability of amino acids to be use in making the recombinant protein.