Protein expression and purification

Basic principles

The media of choice for the growth of E. coli cells containing an expression plasmid are LB medium and its modifications, 2x YT, or Super Broth, each containing the relevant selective antibiotic(s). Initially, it is advisable to try expression in all three media in parallel, and to do a time course analysis to monitor growth and expression after induction. Striking differences between the level of expression in different media and at different times are often observed.

Poor plasmid maintenance in the cells can lead to low expression levels. Ampicillin is an unstable antibiotic and is rapidly depleted in growing cultures due in part to the -lactamase secreted by resistant bacterial cells.

It is important to check plasmid levels by plating cells from the expression culture on plates with and without ampicillin. If the stability of the expression construct is a problem, the cultures should be grown in the presence of 200 µg/ml ampicillin, and the level should be maintained by supplementing ampicillin during long growth periods. Alternatively, the cultures may be grown in the presence of carbenicillin, a more stable -lactam, at 50 µg/ml (see Antibiotics in the DNA section of this Protocols and Applications Guide).

Small-scale expression and purification experiments are highly recommended and should be performed before proceeding with a large-scale preparation. In many cases, aliquots of the cells can be lysed in a small volume of sample buffer and analyzed directly by SDS-PAGE. The use of small expression cultures provides a rapid way to judge the effects of varied growth conditions on expression levels and solubility of recombinant proteins. Expression levels vary between different colonies of freshly transformed cells, and small-scale preparations permit the selection of clones displaying optimal expression rates.
The method used for induction of protein expression is dependent on the plasmid vector and E. coli strain used. Protein expression can be induced by a raising of the incubation temperature or by the addition of an inducing chemical such as isopropyl-b-D-thiogalactoside (IPTG) to the culture medium. Details of induction methods and the plasmids they relate to can be found in standard molecular biology texts (3, 4).
To optimize the expression of a given protein construct, a time-course analysis by SDS-PAGE (see Protein analysis: SDS-PAGE) of the level of protein expression is recommended. Intracellular protein content is often a balance between the amount of soluble protein in the cells, the formation of inclusion bodies, and protein degradation. By checking the protein present at various times after induction, the optimal induction period can be established (see figure Time-course analysis of protein expression).
Time-course analysis of protein expression
We recommend the colony-blot procedure (see Preparation of colony blots and figure Colony-blot procedure) to identify clones expressing a protein and to distinguish semi-quantitatively between expression rates. This can be an advantage for selecting clones after transformation, since freshly transformed colonies may differ significantly in their expression rates. Using this method, colonies subsequently found to be expressing proteins at rates as low as 0.1 to 0.5 mg/liter are easily distinguished from colonies that do not express protein.
Colony-blot procedure
X
Cookies help us improve your website experience.
By using our website, you agree to our use of cookies.
Confirm