Regarding first part of your question, I too had induction issues but i got overcome by lowering the induction temperature to 18 degree with incubation time upto 12 hours with 0.1mM IPTG. I used pET28a(+) expression vector. I suggest you to check the expression at low temperature and low IPTG concentration. Regarding second part of the question I have no idea.

Good luck.

Dear Devi,

I don't know what to say about the first part.

But for the second part, where are you loosing your protein? Is your protein coming into flow-through? Or is it aggregating?

If it is there in the flow-through, then check the filter membrane, you might have a leaky membrane. In that case, changing the membrane to a new would help. Or try some lower cut-off membrane like 3 kDa.

If there is aggregation happening, you should try changing the solvent conditions (e.g. changing pH or salt concentration). That might help.

Best,

Jogender

For the first protein, you could try different vectors and bacterial strains.

For protein 2, maybe it´s precipitating during concentration with Amicon filter. Check the sample for precipitates while you´re filtering it.

For the first you use low IPTG starting from 0.05 induced at different OD600 like 0.4-0.65 and incubate at 16C overnight or for 18hrs or 25C for 14hrs. I do hope it will work. You can try the same vector in Ogama and Rosseta strains of bacteria. 

For second part. you try to get a new amicon filter tube of 30KD. dont worry, your protein will not flow with solvent. For first time use, after fitering, again add the same flow through and do centrifugation. You can also increase the NaCl concentration in your protein solution which will help you to avoid precipiation of your protein during conecntraion step. 

Dear Devi,

For you first protein, as some told you, I would recommend you to make an induction at low tempreature overnight (lower to 18-20°C for 1 hour, then induce at DO around 0.6-0.8 overnight with 0.2 mM IPTG). Also, low yield can come from rare codons in your sequence: using Rosetta cells would improve greatly the yield if your problem comes from this.

For the second protein, needs more info, notably the pI of your protein and pH of the Buffer, the need of cofactor or Ions, the salt concentration used in the buffer, etc.

Hi Devi,

For your first questions, I suggest you try

(1)more mild induction condition: 0.2mM IPTG at 16°C overnight; in addition, as your protein is very small, you'd better also try more harsh condition: 1mM IPTG at 37°C 10h;

(2)reconstruct your gene to a vector with big tag fusion, eg. GST, MBP, SUMO, which can increase the solubility and refolding of your protein  

(3)if your protein is eukaryotic, try the yeast expression system or insect cell system

For your second question, i wonder whether your protein was purified in the elution buffer (250imidazole) or not? Did you check it with sds-page? Because there's one possibility that your protein was expressed as inclusion body, which can be detected by WB with whole cell, but can't be purified with supernatant.

If you checked that protein is available in elution buffer and the tube is good, your protein may be precipitated or digested. Did you see some precipitant in the bottom of your concentrator tube? If so, you can try add some protease inhibitor and glycerol in your buffer.

Good Luck!

Hi Devi

For your second question, its possible that the protein is sticking to the filter membrane of your concentrator..if you have already checked the flow through, maybe you can use a mild detergent or increase the salt concentration of your sample to avoid its sticking to the membrane.

Hope this helps

Hi Devy,

If you don't see any induction for protein 1, you should as well check the promoter sequence. Most sequencing primers won't sequence the promoter region, but everything downstream. If you have a mutation within the promoter/operator, this may influence induction (and could explain that you don't see any induction at all, which is rather untypical). If your protein is toxic to the cells, you may have selected for clones, which harbor mutations in the promoter region (which would completely avoid expression).

Hi, 

Protein 1: we tend to follow a plan of action when initially expressing proteins in E. coli.

1. Test different media i.e. 2 xYT, LB, TB (TB is pH buffered)

2. Different temperatures: RmT-37C

3. Time of induction and OD at time of induction: It is not clear from your explanation how long you left your induction for and at what OD you induced. We carry out a time course on induction and take samples at say, 30 mins, 1hr , 2, 4,  6 and o/n. We have noticed that over induction can lead to loss of protein on occasions especially form inclusion bodies

4. Vary IPTG concentration. 

5. If you have other plasmids with a different promoter then it might be useful to try them: pET vectors contain strong promoters and sometimes its best to use a different promoter /cell line combination.

We would normally try points 1-3 first using IPTG at 1mM final and see what we get, usually at 37C initially. Initial induction OD of 0.4-0.6 with perhaps 1-2 different media over a time course. We also test for solubility analysing both cytosol and pellet of lysed cells

Other points:

- Your product may be toxic to the cells and you may notice a lack of growth in the culture upon induction. You may need tighter control of expression. You may also notice that when plated onto agar the colonies grow slower than expected.

- Codon usage might be a problem: there are strains that can be used for this see BL21 RIPL etc

As for protein 2: I would test the concentrate and filtrate for your product and compare levels to the before concentrated sample. It may indicate that your product is sticking to the sides of the vessel or it may be aggregating. The more you concentrate a protein the greater the chance of association (aggregation). There are additives that you can add that may prevent this happening providing that they do not interfere with other downstream processes.

good luck

wait a second, how can you not get induction by IPTG if you have confirmed expression by Western?