Standard complex media like LB that is routinely used for cultivation of E.coli and performing various experiments including protein expression has several serious limitations. For example, lactose contamination increases background level of protein expression, and therefore may interfere with cloning and expression of toxic proteins. Conditions for protein expression on LB require optimization. Can you suggest the media receipt that lacks of background protein expression and provides high level of protein expression in E.coli with minimum optimization efforts?
Hi Dimitry,
using an E.coli strain with pREP repressor usually worked well for me. It pretty much abolishes "leaky" expression of the protein of interest.
E.coli BL21 (DE3) RE4 would be an example.
Addition of 1% (w/v) glucose when growing cells for expression is also favorable, since E.coli doesn't use lactose as long as glucose is present in high concentrations.
Best Kai
Dear Dimitry,
Normally Autoinduction medium works fine. We have a publication on this however there is another from Studier et al. also describing autoinduction medium.
Our first choice however is TB with low temperatur induction (20°C) for at least O/N.
Fresh transformants and working under suppression with glucose up to 1 gram is possible to reduce toxic effects. Also in the cloning plates you have to add glucose if the protein is very detrimental for cell growth
Good luck
Joop van den Heuvel
Dear Dimitry,
For some proteins it is minimal defined medium M9 that works too and for induction you can use 1mM IPTG , as you do with standard protocol.
Dear Dmitry,
This recipe works fine for our lab.
Good luck!
http://www.chem.tamu.edu/rgroup/barondeau/documents/autoinduction.doc
As others have said, leaky expression probably has more to do with your plasmid than the medium. pLys has worked in our lab expression of some proteins.
Usually, I use LB but many colleagues use Terrific Broth in which you get a faster growth and more important biomass but keep in mind that you will also express more efficiently many protease genes. the best is to try different media in small volumes and different times of induction,
Colette
agree with all others! Yes, auto medium works great in our lab. Here is the recipe and protocol. I would try BL21-DE3-pLys cell line for toxic proteins as well . Another system you might want to try is the Sumo-his double tag expression vector, which helps expression level and most importantly helps the protein solubility in E.coli. Besides, Sumo protease is the most efficient and lowest cost protease to remove fusion tags (compare to Thrombin,TEV, and EK). Here is the link for details; the expression vector (with sumo-his double tag vector) is free when you order Ulp1 SUMO Protease. You can use BL21-DE3-pLys cell line with this vector as well!
http://www.enzymax.net/sumo%20protease.htm
ZYM-5052Medium：
1%Tryptone,1% Yeast Extract,
25mM Na2HPO4 25mM KH2PO4
50mM NH4Cl,5mM Na2SO4
2mM MgSO4 , 100uM FeCl3
0.5%glycerol 0.05%glucose 0.2%lactose
1.Make Basic medium: in 960ml ddH2O add the followings:
Tryptone 10g
Yeast Extract 10g
Na2HPO4 .7H2O (MW=268) 6.7g
KH2PO4 (MW=136) 3.4g
NH4Cl (MW=53.5) 2.67g
Na2SO4 (MW=142) 0.71g
MgSO4 .7H2O (MW=249) 0.492g
Glycerol 5ml
Autoclaved the medium for 30 min.
2. 2M (36%) Dextrose (glucose): MW=180.16 (make 500ml) (720x)
180g in 500ml ddH2O
Autoclave slow exhaust or filter (0.22um filter) sterilizes the solution.
20% Dextrose: weight 20g and add ddH2O till 100ml (400x)
3. 5% Lactose: (make 100ml) (25x)
weight 5g and add ddH2O till 100ml
Autoclave slow exhaust or filter (0.22um filter) sterilizes the solution.
4. 1. 0.1 M FeCl3-6H2O (MW: 270.3): (make 500ml) (1000x)
Weight 13.5g FeCl3-6H2O and dissolve in 0.1M HCl (which is 100 fold dilution of concentrated HCl):
How to prepare:
Add 5 ml concentrated HCl into 400 ml ddH2O, stir to mix. Then weight 13.5g of FeCl3-6H2O and stir to mix. Add the ddH2O till 500ml. Note: Do not autoclave this, use filter unit.
Protocol for auto-induction:
Complete medium: 1L
Add 1.4ml 2M Dextrose (or 2.5ml 20% Dextrose), 40ml 5% lactose and 1ml 0.1M FeCl3 into medium to the final concentration.
Auto-induction
1) Pick up a single colony from a fresh AMP plate into 10 ml of LB/AMP 100 ug/ml for overnight at 37oC.
2) Inoculate overnight culture 1:100 into ZYM-5052 +100ug/ml of AMP, Culture at 37oC for overnight.
Note: You can change the ratio of glucose, lactose and glycerol for different proteins. You can also change the culture temperature to 20-30 oC. You should always use small volume of auto-induction medium to find the optimized condition for your recombinant proteins.
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