Sasa, although I have not used these vectors before, I wanted to ask if there is a consensus Shine-Delgarno sequence 5' of the ATG start codon on your inserts. 

Yes, here is from the manual:

"pDEST™15 and pDEST™17 are N-terminal fusion vectors and contain an ATG initiation codon upstream of the GST and 6XHis tags, respectively. In each vector, a Shine-Dalgarno RBS is included upstream of the initiation ATG to ensure optimal spacing for proper translation initiation in E. coli."

The vectors are created for protein expression in E. coli.

 Are you trying to express a membrane protein? In that case it might just not work.. snd no-one can really explain why. Do you have other pDEST vectors in house? you could try MBP or Trx fusion instead. Sorry for the discouraging advice, but it seems you are doing everything correctly and you might need to consider that there is no or only weak expression. Check with Western Blot to be sure!

Have you tested insoluble fractions? Any chance these particular fragments just aren't soluble, but are being expressed, etc.?

Hey Tobias and Jesse, thank you for your answer. It isn't a membrane protein. I don't have any other pDEST vectors, so I will probably just reclone everything into completely different vectors (ugh).

Jesse, I checked with Western Blot and I can't detect any protein in whole cell samples after induction. So I guess it just isn't expressing or it gets immediately degraded after expression. I never had a case like this before. From all predictions and similar domains/folds the fragments should be soluble.

Hi Sasa. Your approach still does not rule out Antibody problems. Have you tried pulling down using Nickel NTA beads  followed by SDS PAGE and silver staining (and probably a western too) to distinguish between No expression vs very low expression?

Hmmm. What are you using for your Western Blot (antibody-wise)? Also- are you using Rosetta/are your proteins eukaryotic?

Hey Priyadarshan - you are right! The problem was an extremely low amount of protein. I repeated my WB after pulldown (still nothing visible on beads by SDS-PAGE) and seems like there I couldn't detect the low amount of protein previously. Now I can see there is no expression of His-tagged protein (for sure, control is nicely visible but no bands for the after induction samples), but for GST-tagged I can see thin bands (I really hope it's the protein, sizes look good!). So I have very low expression with GST. This is not so great, because I need a lot of protein, but certainly better than no protein!

Jesse, it is an eucaryotic protein, but I am using different E. coli strains, some of which are optimized for eucaryotic protein expression. It is not the perfect system, but it has worked well previously. If it doesn't work, I will try other systems. Do you have some additional advice on that topic? Do you have particularly good experience with a certain strain (or insect cell, yeast...)?

Hi Sasa. I am glad that you were able to move forward with your project. If possible, could you please share which protein you are trying to over express?

Hello Sasa,

If you are cloning in pDEST15/pDEST17 vector then it will contain  araBAD promoter and you have to use L-arabinose for inducing the expression of your of gene of interest.

Hop it will work.

For more information go through this link.

https://tools.thermofisher.com/content/sfs/manuals/ecoli_gateway_man.pdf

Thank you,

sai..

Hi Sasa, any chance you can send me an aliqute of pDEST15 and pDEST17? I want to start to use them to express proteins but are very expensive 😐. I can pay for the shipping. Thanks!!!

It has been long time after this question but ı ve faced the same problem and solve it by using bl21-aı strain and l arabinose induction in low temperature (19 •C)

I used BL-AI strain with pDEST17, induce at OD 600 : 0.42 by 0.2% L-arabinose, and incubate overnight at 24 degree C. Why is it still no expression of my target protein? Actually my protein is small size ~16 kDa and not toxic

Dear ha, did you collect a sample from your culture every one hour.. you can decrease the temp. Optimal induction time was 4h for my protein. For sure it differs form protein to protein but ı dont have any idea about 24 h induction ı did not perform. Even if you did not see anything in commassie you should perFORM WESTERB blot good şuck

Thank you so much, Gulden! I tried some other conditions, 2 hours, 3 hours of 0.2% L-arabinose induction, and 3 hours of 0.1% L-arabinose induction. I also tried to use another strain, the BL21-DE3 and induce with IPTG. All induction were conducted at 37 degree and induce at OD600: ~0.45. Still no band of my targeted protein. I successfully expressed that protein with GST tag with high efficiency at first trial. I used Gateway cloning to replace into His tag. I also sequenced the inserted pDEST17 to confirm, there is no problem. I tried to do His-tag purification, I got my band, but it is very weak along with other stronger nonspecific bands. I'm gonna keep trying to find out another better induced condition.

Dear ha, ıt may form inclusion bodies and stay in pellet or your protein is toxic for that strain. if you get a thin band you can see it in WB. or you ll try to get it from pellet with spesific enzymes (as far as ı remember you can add lyzozyme to your lysis buffer). If it is expressed in GST that means GST gave solubility to your protein. Induction with IPTG is meaningless ı thınk cause there is no lac operon in pDEST17. it is recommended with L arabinoz. you can also try his-MBP tagged pdest566. MBP will give solubility and you can pull it wit Ni Nta beads. good luck again.

and ı suggest you to decrease the temp to 19 degree C. stronger non spesific bans are not non spesific as well ıf you did purification. they are degraded and shorter forms of your protein.

Thank you so much for valuable suggestions to me. It is reasonable that stays in the inclusion body as you explained by comparing with GST case. However, I ran the gel with both the bacterial culture and soluble lysate. I thought that could show if my protein was induced in bac.culture sample, not in soluble lysate, meaning it is in inclusion body. I will check the precipitation after centrifugation to confirm, perhaps the bac. culture is too little to check. I hope my protein sit somewhere, even in inclusion body. I also tried with 19 degree, but it doesn't work. My targeted protein is about 18kDa, but the nonspecific band is above it. I also tried once that added 0.1% glucose with 0.2% L-arabinose as some book said. Nothing works. And all after adding L-arabinose, the bacteria grow really much slower than comparing to not inducing sample. I observed it from that I load the same volume of bac. culture but there is always the total protein from induced samples more faint than not induce, but also possibly due to my technique.

This is one picture with loading bacterial culture ( 22ul for each well). 1st lane is not induce, then 4 lanes of different condition at 37 degree, then not induce , following 3 different induced condition at 19 degree C. I have another gel with soluble lysate, not shown here, the bands are stronger but not my target. The second band of ladder from bottom is 15kDa, and my protein is estimated above that

I tried to induce with IPTG once, just because, when I searched pDEST17 in introvigen as the origin of my vector, they give information that vector is inducible and induced agent: IPTG. I don't understand that much, then I still tried. It also does not work. Anyway, I will check my insoluble that is important point.

Thank you so much for your help, Gulden!

https://www.thermofisher.com/order/catalog/product/11803012

hi ha,

ı got the point it states IPTG as induction agent but when you check the manual it recommends BL21-AI which is suggested to be induced with L-arabinose. how about your experiment? Were u able to find the BL21-AI strain ?

https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2Fecoli_gateway_man.pdf&title=RS4gY29saSBFeHByZXNzaW9uIFN5c3RlbSB3aXRoIEdhdGV3YXkgVGVjaG5vbG9neQ==

The expression efficiency is still very low by comparing with not induce. In not induce sample, I also see my target band. I induced by 0.2% L- arabinose + 0.1% glucose overnight at 30 degree. And one more problem is the target protein mostly sit in the inclusion body. My protein is both low expression and insoluble. Anyway, Thank you so much for helping me a lot!

How about the results? Od600: 0.45 is low is it not? Optimal od is btw 0,6-0,8 in e coli

Thank you for helping. I terminated this experiment. The target protein mostly stayed in the inclusion body. I don't want to denature and renature for purification because the downstream exp. is sensitive. I think I'd better modify the vector.

Ok good luck