小中大OK, the point you are making is about optimization. Well, the optimization is a very hard work and you must be extremely careful and patient. The most important and troublesome is that it have something about luck pretty much.
According my experience, a general rule of optimization is that:
First, you have to make sure that the gene is corrected inserted into the vector. Namely, make sure the sequence of your recombinant plasmid is correct;
Second, if one competent cell such as XL-1Blue, does not give good expression after extensive optimization, please try others such as BL-21;
Third, if the protein is mainly in the pellets, you can extend the extraction time and strength;
Fourth, if still difficult, express the protein in fragments;
Finally, try different vector systems---different vectors.
For detailed optimizing work, You have to express the protein as following expressing system:
OD:0.8, IPTG:1mM
37 degree inducing for 2hr---with/without IPTG;
30 degree inducing for 3hr---with/without IPTG;
30 degree inducing for 6hr---with/without IPTG;
22 degree inducing for 8hr---with/without IPTG;
14 degree inducing for O/N---with/without IPTG.
Then, compare the results with each other and make sure the best induce condition and further optimize the express based on the best condition come from above work as following system:
OD: 0.5, IPTG:1.0mM---with/without IPTG;
OD: 0.8, IPTG:1.0mM---with/without IPTG;
OD: 1.2, IPTG:1.0mM---with/without IPTG;
OD: 1.5, IPTG:1.0mM---with/without IPTG;
OD: 1.8, IPTG:1.0mM---with/without IPTG;
Then, compare the results with each other and make sure the best induce condition and further optimize the express based on the best condition come from above work as following system:
OD: the best value from above work.
IPTG: 1.0mM;
IPTG: 2.0mM;
IPTG: 5.0mM;
IPTG: 10.0mM;
If you still can not get the good protein, you have to change to other medium, such as from TB to LB or from LB to TB, to see the results.
Though it's very hard, it can give you good protein for more than 90 percent of proteins.
The above is the stradegy used in our lab, I hope it can help you out.
FOR YOUR REFERENCE ONLY
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