Crystallization quality protein for X-ray diffraction is most certainly of a good enough purity and concentration for Cryo-EM, although still Cryo-EM demands an intrinsically large protein or protein complex by molecular weight. Cryo-EM is not going to see the existence of a 20 kilodalton protein even if it is 100% pure, crystallizable , and at a reasonable concentration. It is just too small for Cryo-EM to see.

Escherichia coli is the best workhorse for producing a vast amount of protein. The difficult part is fishing out your protein of interest from all the other proteins in the cell. This usually requires affinity chromatography. Size Exclusion chromatography for protein of interest yields exceptionally pure protein, but it also reduces the concentration because although your protein is purer you do lose some of your protein with the technique.

There are cases where E.coli would not be the best, mainly if the protein you are studying should be glycosylated. E.coli doesn't have a Golgi organelle capable of glycosylating your protein.

Thank you Adrian,

I realize this, but it has been suggested to me that because the amount of sample needed for cryoEM is so much smaller than X-ray or NMR, it may be advisable to engineer a tagged protein and integrate it in single copy in the chromosome of mammalian cells via CRISR/Cas and perhaps capture it with magnetic beads. Any thoughts on this?

Thanks,

Dave

There are plasmids capable of transfecting certain mammalian cells, even some plasmids can even transfect Plasmodium falciparum parasite. It would probably be much easier clone your gene using polymerase chain reaction combined with either restriction enzyme digestion and ligation or Gibson assembly into an inducible plasmid. Then you can transect that plasmid into a stereotypically well expressing mammalian cell like HEK293. During PCR is when you can add the tag like a Sumo tag. In fact some plasmids come with tags near the protein insertion site and so you use molecular cloning methods to ligate your gene into the tagged plasmid.

Once you have your plasmid with your gene in the multiple cloning site you can just store it at -20°C and it will stable for a long time. To be sure your plasmid is correct you have to sequence it with primers about the multiple cloning site where your gene is. To do CRISPR is re-engineer the whole genome of an organism.

Hey, thanks for the advice. Sorry I misspelled your name.

Dave

I still think that chromatography techniques are the best to use to get a pure protein in the end. If you are not tagging your protein of interest you usually think of a 3-step purification to get your protein pure (but more steps can be required). CEP strategy: Capture usually on an ion exchange resin, Enhance often using an opposite charged ion exchanger compared to the first step or a hydrophobic interaction resin and finally the Polishing step using a size exclusion step.

Purifying a tagged protein or an antibody is usually simpler often first an affinity chromatography step followed by a size exclusion step (sometimes you may need a ion exchange step in between).

Hi David,

If you are interested in the latest technologies, check out in vitro protein expression. Is typically done in small scale, but if you need more protein you could scale it up. Purification requires less effort and you easily change the gene you want to express. Think there are publications where they add the enzymes needed for glycosylated proteins, unfortunately no refs at hand.

Best regards,

Anders

Hi David,

In the field of membrane proteins, various novel technologies have been developed to purify and produce high-quality membrane proteins with high purity, such as liposome technology, virus-like particle technology, and nanodiscs technology. These tools can keep membrane proteins in their native state to determine their 3D structure and study their functions both in vivo and in vitro.

Dear Roger,

Can you provide any references on these membrane technologies or leads to companies that market them?

Thanks,

Dave

Dear David

In my opinion, also if it was developed already about 10 years ago, the Expi293 expression system, is one emerging mammalian system which is able to combine properties as high cell density and the high expression yields that until few years ago were restricted to E.coli , Pichia pastoris and baculo with the amazing ability of mammalian to produce PMT (especially S-S bond formation) and is something that may allow to produce a lot of uncharacterized mammalian soluble proteins (as well as monoclonal antibodies) in folded form and high yields also with out producing stable clones (transient trasfection) and the protein are released on the cell surtatant and therefore a single IMAC purification step is enough to reach high purity level.

Thermofisher developed also a Expi 293 Gnt- strain for homogeneuos glycosilation which is usefull for X-ray.

The system is quite expensive, but also the yields in some cases are amazing and several alternative cheaper transfection agents are coming..

If you are interested you can find a simple video of introduction to this system on the following link

https://www.blogger.com/video.g?token=AD6v5dyTuEwDXD2ztCnLIFHkG06cCp1jaRuUHOGFtHdiTFnj-yS_TyCyPqzvi6RogJaSP_sXqbJnMDIYplkXODpfOc21BETPPh74J7FJuwMPxHW4X79mURcAl7-V0ApA8BQrGUAgcSc

on my blog ProteoCool.

In the last 10 years i had also the opportunity to wark with 2 other interesting systems as:

1) Brevibacillus expression system

High-level intracellular expression of heterologous proteins in Brevibacillus choshinensis SP3 under the control of a xylose inducible promoter - PubMed (nih.gov)

which may be an interesting back-up host to use for those microbial proteins that E.coli is not able to produce

and

2) E.coli BL21 Delta TolR (or other) hypervesciculating strain as tool to produce, recover and purifiy those protein containg S-S bonds (as for example fabs) from the sister lume instead the bacterial periplams.

Probably you are already know those systems and any of those are really the latest technology.

I hope that some of those advice can be usefull to you!

Ciao

Manuele

Hi Dave,

Not sure who you are looped in with, but I collab. with the folks in Frederick at ATRF for cryoEM. Great resource: https://ncifrederick.cancer.gov/about/atrf/default.aspx

In addition, the NCATS crew is another resource of minds to pick for ideas.

Best,

Thomas