Did you look for the protein in inclusion bodies? Double check the cloning put the protein in frame with all the required expression components (promoter, Shine-delgarno etc...). Could even try RT PCR to determine if there is any gene expression.

I checked for inclusion bodies, because I ran SDS-PAGE for whole cell. cloning had no issues, because I have used pET plasmid many times and no issues so far with small proteins. Also checked my sequence multiple times by sequencing, and still it was good. I have not used RT PCR for bacterial protein expression yet. Is there any good reference I can follow? Thank you for your comment.

Hi Kim! I did express high MW proteins is in E.coli using Rosetta strain. Expression could fail for a lot of reasons. How do you check for your protein expression? I mean do you separate soluble from insoluble by extraction or you boil the whole cells? If I remember well Rosetta strain bear extra tRNAs for maximizing low frequency codon usage in E.coli. Check if the codon usage in your sequence is optimized in that particular strain. I think they have various strains.

If time to work out the trick is precious, then why not to look for other system. I am not sure you will get mg quantity from a mammalian expression system but there might be. Instead I suggest you express it in Pischia Pastoris (A type of yeast used for expression of proteins). You can order it from Invitrogen together with the expression plasmid. I haven’t tried myself but a colleague did express its protein in Pischia Pastoris.

I've never done RT-PCR on bacteria before, but I imagine a simple Trizol purification and the RT kit from Invitrogen would do the trick. But this isn't the greatest idea to be honest. You could certainly try this in parallel with other troubleshooting, but don't waste too much time here if you are confident with your cloning.

The fact that you didn't see anything in inclusion bodies is troubling. I assume you checked your IPTG, growing temperatures, induction times, antibiotic concentration/fidelity.

Until you know the problem, I wouldn't worry about yield. Do the Western Blot. The great thing about E.coli is that you can scale up if need be, but you can also optimize this procedure quite a bit too. The results of the Western Blot should help guide your decisions.

Hi Younghoon,

For protein hard to express, i suggest you to clone it with a GST tag in N-terminal, such as the one you can found in pGEX vectors, and/or to try the SoluBL21 strain from Gelantis for the production. I had very good yield with this procedure.

But if you need few amount of the protein you can try the human cells, the folding of the protein should be better.

As underlined above, there are several problems with heterologous expression.

assuming that the DNA construct is correct one can list :

1- The nature of the protein itself is the tricky point. The protein may require assistance to fold (chaperone apparatus typical of the organism of origin) otherwise you will get inclusion bodies (which seems not to be your case).

2-do you observe that cell growth slows down dragstically after induction ? Observe your cells with a microscope after 3 hours induction and titrate for a possible toxicity of your protein.

2- under-represented loaded tRNA (rare codons in E.coli) lead to ribosome disassembly and translation stops giving a population of shorter proteins which people often missinterprate as proteolysis patterns in SDS-PAGE.

3-expression conditions : time of induction, temperature of induction, plasmid copy number (pET is fine actually). For large proteins cells might require more food (consider adding extra glucose in your LB medium) it increases yield.

( I will not discuss membrane protein problems of course...)

You have several option to deal with rare codons problems. Roseta is a good option but all codons are not represented in that strain. If you are rich optimize your gene for E. coli by ordering a synthetic gene. Or change of system as suggested. The easiest way is to switch to P. pastoris. Can be installed quite easily in a modest lab. Another way which will not give you a high yield but very good quality eukaryotic proteins is insect cells (but required an equipied lab; why don't you collaborate ? the best way for efficiency; we cannot do much alone but shearing with others leads to great achievements).

That is what I have at the top of my head right now but there are many other issues.

Take care

a last idea :

maybe you already did, but explore the occurance of rare codons in your sequence with :

http://nihserver.mbi.ucla.edu/RACC/

You can check the amino acids composition of your proteins and try BL21 DE3 RIL or RT, I have good results in these cells.

Hi kim,

I can see there are already many gr8 suggestions. I would say first check your construct and see if your gene of interest is in frame. Then I have had some issues of expression with toxic proteins. So if your protein is a phosphatase or toxic protein, i would certainly recommand to use AI cells for expression and this can make huge difference. Just give a try you will know. Gud luck.

Raj

What kind of protein are you trying to express? For example, expressing a membrane protein in E. coli is very difficult. Also, if your protein is a multidomain or unstructured protein, it may be proteolyzed. Do a Western blot to see if your protein is being expressed at all and whether it is being degraded.

I think Raj's comment is very valid: check the construct by sequencing. At the same time, use 10-20 clones after initial ligation to setup small scall expression and look for differences. Sometimes the cloning itself, especially when starting with RT-PCR, can result in frequent frame shifts or deletions. Use, if possible, a verified cDNA from a commercial source or a collaborator.

Hi Kim

I think that you should change another host strain. I recommend you B. subtilis cell with pHT expression system (pHT01 or pHT100 is good way) . For more information about expression in B. subtilis cell, you can contact to my Proffessor - Dr. Hoang Nguyen - via e-mail: [email protected]

About expression in B. subtilis cell, I used to expressed a large protein (about 110 kDa) and it worked very well.

Good luck Kim

We've expressed large proteins in E. coli to good levels. However on the whole the larger a protein is the less likely it is to express in E coli.

You do need to check whether you have any expression at all. Running total extracts and looking for a band isn't the best way of analysing expression, it depends where your protein runs but you can often have yields of 2-3mg/l and not be able to see the induced band against the background.

We always do a (Ni-NTA) pull-down of the soluble and if necessary the insoluble to check for expression, because we are high-throughput we tend to always have a His tag even if there is a solubilising partner like MBP (for the record generally GST isn't great as a solubiliser!) and use magnetic beads. However pull-downs with Ni-NTA agarose or the Qiagen spin kits also work well. We then run gels. We test 1ml of culture and if you then can't see a band on the gel then its probably a non-starter.

If you see no expression at all switching expression systems may be the best option, we use TRIEX based vectors and would probaly go into insect cells next. If you see insoluble expression or low level soluble expression then rescue strategies should include fusion partners, changing expression strains and induction conditions.

I can see some really good suggestions already given. One other way is actually a tougher one but with much higher percentage of success, specially for proteins with high post-translational modifications.

Try to express and purify in baculovirus system if it is possible in your facility. It is relatively expensive but in that way you could get the protein with almost all post-translational modification done as it is cultured in insect system. I'm giving you one link to understand how the system work, but there are many other ways to do that.

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Protein-Expression-and-Analysis/Protein-Expression/Insect-Expression.html?s_kwcid=TC|12127|%2Bbaculovirus%20%2Bexpression||S|b|9458605867

Hi, here are already a lot of good answers! Just in case that you have a membrane protein, you could use the BL21 derived Walker strains C41/C43 or LEMO21 (http://www.ncbi.nlm.nih.gov/pubmed/22858868). Good luck!

First of all you should check that your insert is in the open reading frame, I would suggest that you use the pRSET plasmid and the E. coli BL21 (DE3) pLys E, the bacterial strain with plasmids using the T7 promoter have already possessing a plasmid encoding for lysozyme that helps reduce basal levels of expression of heterologous genes due to controlling the T7 RNA polymerase very useful when you have toxic proteins, or you can use the pGEX, where you also get lots of protein.

It is also important to check whether the protein itself is not toxic, for example, decreased cell growth, if so will lead to less time required, and adding glucose to the culture medium to minimize toxicity. Good Luck!!!!!

I agree with the previous speakers, sequencing of whole construct is a basic thing to do. The question is how much do you know about this protein? Does it contain any introns E. coli is not able to process properly? Does it require posttranslational modyfications?

If the activity of the protein is the problem, you should see after induction rapid decrease in growth speed. Did you try to express in lower temperature? In our lab huge proteins were expressed in E. coli cells with good yields in 16C.

First of all my question is have you prepared cDNA for expression? If yes first check orientation of correct frame of gene by using one primer against vector and another primer against specific gene. If it is ok next go for SDS-PAGE with control. And check for any new products by comparing with control. If you find any new products other than 120kda that means the protein is prone to degradation.