Hi there,

LB is definitely containing free glucose which is the issue if you want to use lactose as inducer (glucose being a strong repressor of lac promoter). Studier's auto inducible media is what you need: it allows producing biomass on glucose then switching to glycerol and lactose as the only available carbon sources. For info:

https://openwetware.org/wiki/Lidstrom:Autoinduction_Media

"In order to figure out the optimal condition for induction

with lactose, concentration- and time-dependent induction

was investigated. As shown in Fig. 1" have been said in your reference.

You have to check different con of lactose to fined optimum induction.

Good luck.

Hi there,

LB is definitely containing free glucose which is the issue if you want to use lactose as inducer (glucose being a strong repressor of lac promoter). Studier's auto inducible media is what you need: it allows producing biomass on glucose then switching to glycerol and lactose as the only available carbon sources. For info:

https://openwetware.org/wiki/Lidstrom:Autoinduction_Media

Hi again,

Lactose has actually no solubilizing effect on protein aggregates but it allows to prevent the misfolding of protein by slowing down transcription/translation process compared to what IPTG does.

Hi,

Factors like size of the protein, induction temperature, co-expression of chaperones will impact protein solubility. If your protein is eukaryotic in origin then you can add PTMs as an additional parameter. E. coli, though the most robust heterologus system for protein expression, often shows this problem. You can check protein solubility prediction using software PROSO II. This works considerably well for small proteins of around 35-50 kDa. But if all the above parameters are considered and still protein is insoluble then using E.coli strains like origami (for proper disulphide bridge formation if your protein has disulphide bridges) might help. Only empirical trial and error method works with protein expression and purification. If you have access try insect cell line if your protein is eukaryotic in origin. Yield might be highly compromised.

Hola, Tryptone and yeast extract contain lactose in low amount but lacY is able to take all the molecules from the media towards inside the cell, so the expresssion is induced from the inoculation stage, so you have a "quasi" constitutive expression.. Glucose at low concentration (1-4 g/l) doesn´t repress at all but could aminorate the lactose effect. In my case I always use LB/water 1.1 phosphate (of terrific broth) and glucose 2g/L. starting the culture in 5ml and scaling volume when it is growing. In this case at the beginning the amount of lactose molecules by cell is lower and the induction is light and increasing culture volume with high cell mass helps to control expression. Make a time course expression experiment to check the presence of your protein. But, you know that there are proteins which are insoluble in all the situation specially when the molecular wheight is high or if they are heterologous, for instance mammalian origin. Buena suerte

hi,

we usually go by the recommendations given for the vector. however, we do optimization in 5 ml cultures with 0.5 to 2.5 mM IPTG and also for 5 hours and 8 hours keeping the temperature constant at 28 deg. if u feel there is high inclusion bodies you may reduce the temperature from 28 to anywhere till 20 ..

Hi,

I agree with Krishna kumar Ganta. Try to lower temperature. In my case, I cool the culture on ice, then add IPTG (depending on the protein - final concentration 05.-1.0 mM at OD600 0.4-0.8) and continue the culture overnight at 21C. It works nice with my human enzyme. If you will lower temp. you will get less cells so less recombinant protein of course, but bacterial culture are cheap, fast and you can easily scale it up :) The goal is to get an active, soluble protein :)

hello Magdalena,

thank you. the idea behind low temperatures is enabling the protein to have enough time to fold before its pushed outside the membrane as inclusion bodies. proteins in inclusion bodies tend to have misfolded structures and mostly inactive. So cooling will reduce the protein synthesis and let proteins to have proper folding.

I never thought of doing the induction on ice. I don't think I have been doing cold enough temperatures.

Dear Krishna kumar Ganta,

I am happy that I could provide valuable tips for you. I hope that lowering temperature will substantially improve your protein production and you will get it as active and soluble. In addition, I came with an other idea - some colleagues from my department tried co-expression of target protein with chaperones. Here is the link to the article: Article Chaperones Are Necessary for the Expression of Catalytically...

Dear Ung,

I use to induce with lactose for producing soluble HMW protein (Fab) production using E.coli BL21 strain back in the industry in fermentation set up. Please consider the following:

1). Glucose neither should be completely absent or in excess in the media. A very small concentration of glucose is needed to keep the bacteria alive but it shouldn't be enough to carry other metabolic activities. This is the ideal stage to induce with Lactose. The OD at 0.8 doesn't simply means your glucose is very less in the media.

2). The lactose concentration should be between 2 - 4% throughout the induction. Induction will start slowly (normally 6 hours or more) than induction with IPTG. First, you can observe Lactose intake slowly along with the increase in OD. Further, if the lactose conc. is maintained in the above limit as above, the cells will slowly be induced.

3). Lactose uptake rate is very important. It should maintained under particular rate to get your proteins in soluble forms. if you try to optimize the lactose uptake rate, then you can induce most of the protein in soluble forms.

4). Also post induction time also have to be considered. Have said that lactose induction will be slow, but if you allow the strain for long hours after induction the protein may gradually end up as inclusion bodies.

The below reference will be very helpful:

Wurm, D. J. et al. Mechanistic platform knowledge of concomitant sugar uptake in Escherichia coli BL21(DE3) strains. Sci. Rep. 7, 45072; doi: 10.1038/srep45072 (2017).

Wurm, D.J., Quehenberger, J., Mildner, J. et al. Appl Microbiol Biotechnol (2018) 102: 667. https://doi.org/10.1007/s00253-017-8641-6

I hope above points and the reference article will be very helpful for your work.

Best wishes,

Ahkam Saddam S.H.

I have successfully used autoinduction medium ZYM-5052 to produce 30 – 50 kDa GST-fusionproteins using E. Coli BL21 transformed with pGEX-2T plasmid vector. Recipe for ZYM-5052 was already linked by Dominique Liger

Refererence: F.W. Studier (2005) Protein production by auto-induction in high-density shaking cultures. Prot. Exp. Pur. 41, 207-234

Here is one way to do the cell culture, use with own responsibility :)

1. Start inoculation from a single colony in 5 ml LB + ampicillin culture over night at +37°C under constant shaking.

2. Dilute 1:500 with freshly prepared ZYM-5052 with 100µg/ml ampicillin. Total volume 50 – 200 ml. I left out trace metals from the medium.

3. Culture 1,5 h at +37°C and thereafter at +20°C under constant shaking. Continue ~20h.

4. Harvest the cells.

There are many more things to consider, but I hope this might prove useful for your needs and please ask if you need more information. Good luck!