Science Buddies: "Ask an Expert"

hello, I'm currently nearing the end of my research for a biology practical project, which has been fairly challenging. We had three weeks to research the project of which two weeks are left; however as I knew what I wanted to do I've been researching for about two/two and a half months. The project involves isolation of a membrane protein, specifically OmpC from Escherichia coli K-12. The membrane porin is an antibiotic binding site and is involved in the binding of outer membrane (OM) vesicles carrying leukotoxin to human polymorphonuclear leukocytes and monocytes; at least in virulent strains of the bacteria. OmpC is also part of the Omp super family present in numerous gram negative species. My project is based (loosely) on two papers: one of which reports on how osmotic stress induces Kanamycin resistance in E. coli, because of the proliferation of OmpC (in favour of OmpF) in high osmotic pressure environments. Whilst the other paper, notes a similar effect only the other way around and with resistance to T4 coliphage infection I.e, E. coli in a low osmolarity environment show reduced susceptibility to phage infection.
I want to determine if OmpC does act as the adsorption ligand of the T4 phage to the K-12 cell. To do this I need to isolate the protein, I've found and ordered the bacteria and phage, researched which buffers, proteases/phosphatase inhibitors I need, and the centrifugation protocol I'm going to use.
However, I understand that I need to use chromatography to isolate the protein to a reasonable purity. I've been trying to learn about the process but "as I'm sure your aware" it involves investigating all the proteins in the final OM fraction after ultra-centrifugation, and finding the unique properties such as charge, iso-electric point e.c.t and I don't have time to research it properly before the project is due to start, I've spoken with academics at local university's and they basically think I should abandon the idea, I've spent to much time and money to do that yet though. Additionally, I've realised that I probably don't need it to be 99.9% pure, just free of anything that might interfere with the adsorption process, for example I can't claim with any degree of certainty that OmpC is the phage target if the protein sample contains 50% OmpF.
So this is the part I need advice on, can any one think which chromatograph media I can use to separate the protein from other outer membrane proteins. I'll be using low pressure column chromatography, I know affinity chromatography is especially useful for this kind of thing, but the column packing media is really specific and expensive. the best I've come up with is 'Sephacryl S-100, Concanvalin A Sepharose and what I think is probably the best, Sephadex G-25' from GE life sciences and sigma aldrich; these two media were chosen using OmpC's molecular weight of 38.073kDa but I'm not sure if there right. Also is there any other means of separating specific proteins, such as isopycnic centrifugation (I have access to some one with an ultracentrifuge) or precipitation????
Any suggestion regarding a protocol, chromatography media, information-resources and general advice on procedure or where to look and books to buy, will be massively appreciated!!!!!! I've attached a word file containing some info obtained from the Scripps institute protein calculator, which predicts the features of a protein based on it amino acid sequence this is about the best I've got on any unique features which could be used to isolate the molecule. If there is any more info you require please ask me.

Thanks,

Marc

This is the Scripps institute file.

Hi Marc,

Welcome to Science Buddies. You are doing a very challenging project!

You have obviously done a lot of background reading and you know what steps you need to do to purify your protein. Here are some suggestions that should help.

You didn’t give the citations you are using, but here is an article that gives a detailed procedure for purifying ompC on a small and large scale. The authors include a specific protocol to purify the ompC from E. coli.

http://144.206.159.178/ft/2795/588865/12060697.pdf

The procedure involves first using an Amicon ultrafiltration membrane to concentrate the sample, followed by a size exclusion column using Sephacryl S-200 column. The Sephacryl S-100 would actually be better for your protein because it has a lower exclusion limit, so your protein would elute earlier and be more concentrated. Sephadex G25 has a molecular weight exclusion limit of 5000 Daltons, so your 40 kD protein will elute in the void volume. The G-25 would be suitable for exchanging the buffer, but since the ompC is not included in the void volume, it would not work for the purification step.

The second step is an anion exchange step that would bind the protein and then selectively elute it away from contaminants. I assume that you have found a reference that uses ConA to purify this protein, so this could also be used for the second step.

The primary contaminant in the sample is lipopolysaccharide (LPS). The size exclusion step does not bind the sample at all, so the ompC with its associated LPS would coelute.
ompC is a hydrophobic membrane protein and adsorbs LPS by hydrophobic interaction, so the second step would be required to separate the two molecules.

It sounds like you have access to a laboratory. Please look through the protocol and check to see if you would be able to obtain the buffers, SDS page gels, silver stain, and other reagents used for the purification. The authors of the paper used a chromatography system, which would make the purification faster, but take some expertise to use. Do you have columns available to pack the Sephacyl 100 gel? Do you have access to an Amicon concentrator? Do you have media available to grow 1 to 4 liters of E. coli? What quantity of protein do you need for your experiment?

Please let me know what you are missing and what you have available. With science fair projects, it is frequently necessary to improvise, so I need to know what you do have available to use to make specific suggestions.

Also, please let me know what references you are using and if you have any other questions.

Donna Hardy

Hello Ms Hardy,
Firstly, thank you for taking the time to reply and help. The two papers I mentioned in my first post were "Osmotic stress Induces Kanamycin Resistance in Escherichia coli B23 through Increased Capsule formation - A. Kuzuhiyil, Y. Lee, A. Shim, A. Xiong, (2012); journal of experimental microbiology and immunology, vol. 6 5-10".
And
"Molecular Basis of Bacterial Outer Membrane Permeability Revisited - H. Nikaido, (2003); Microbiology and molecular biology reviews, vol. 67(4)"
However, neither of these contains methods/experimentals on porin purification, there just where I got the basic idea for my project. I do have about 15ish papers with protocols in, which I've been using to construct my own protocol; GE healthcare also have a booklet available as a downloadable pdf file, the subject of which is isolation of membrane proteins. It's called purifying challenging proteins.
I checked today, and unfortunately the lab I'm using doesn't have a amicon ultrafiltration membrane, but I do have access to spin filters which are "apparently similar" although I don't think I'd have time to figure out which unit I would need to use. I think? they were called 'Bio-Rad spin filter tube/columns' they use a centrifuge to force the homogenate through a membrane (again I think ). Although, would it not be possible to collect the outer membrane fraction through differential centrifugation at 100,000g and then use a detergent such as Triton X-100 or tween to solubilise the protein components of the cell membrane; and then collect the OmpC and other approximate proteins using a density gradient centrifugation step?
Other than the ultrafilter, I have most of the other materials. I have as much media as I need and culture flasks, autoclave, ddH2O and an incubator. The media I'm using is a custom broth, based on a lysogeny broth recipe (from this site - http://www.bioprotocols.info/model_orga ... plates.php), only I've tweaked the ingredients to increase the osmolarity and pH. Per litre it contains 1g of lemoco lab powder from Oxoid, 2g of yeast extract, 2g peptone and 3g tryptone, it also contains 0.8 NaCl and 0.2 glucose which raises the osmol's by 3.0.
As for the buffers I'm pretty sure we have Tris-HCl, although could I not use Tris-buffered saline or even phosphate buffered saline???? I also have access to MgCl2 and (I think) to SDS, which is sodium dodecyl sulfate, right??? The EDTA and saline won't be a problem either, and we have SDS PAGE electrophoresis equipment and reagents. I don't have access to a sonificator, although I imagine I can use a pressure cell or lysozyme, do you think this will matter? Also, I don't think I can get the 2-mercaptoethanol. At the moment this is what I have and don't have. Oh and I have access to low and medium pressure chromatography systems, specifically the Buchi sepacore, which has a pressure range from 10-50 bar, and can take a litre of sample at maximum. Or I have access to bio-rad econo-glass columns which I could pack with media and then attach to either a low pressure air pump or apply a mild vacuum via a Buchner flask??? also I need to order protease and phosphatase Inhibitors, so I could really use some advice on which ones I absolutely need or if I can get away with just keeping the homogenate cold (for example).
essentially I want to do the best I can which the budget I have, I can access all of the reagents, like the EDTA and buffers, and the equipment free of charge; and possibly the size exclusion media. However anything that I need to order-in I'll have to pay for, so I'm open to improvisation.
I've now also got the K-12 cells from 4 litres of culture, I pelleted them and then re-suspended them in a PBS with 10% glycerol (and 10% cow serum?) then flash froze them with liquid nitrogen. Although most of the protocols that I've read on-line say to store the cells at -20C > -80C , but as I would have to disturb people to get them in and out of storage at that temperature I've put them in a normal freezer, one of the lab technicians said that would be ok in the short term, once I've lysed them I'll store them at -80C. Do you think that will be alright?
I really appreciate your help with this. Also I'll make a list of the most important sources I'm using and post them soon (most likely tonight or tomorrow)

thank you,

Marc

Hi Marc,

Thanks for your detailed reply about your current resources and for the references you are using. You have found lots of references that will help with this project, especially the guide from GE on purification of membrane proteins.

Here are some more comments and suggestions:

1. Since this is a membrane protein, you are correct; you will need to use Triton-X 100 to solubilize it and prevent non-specific loss to to precipitation and adsorption to surfaces. Always use the smallest surface area possible for each step.

2. Your idea to use density gradient centrifugation to purify the membranes first is a good idea. This article looks like it includes a procedure. You could use the centrifugation step first and then follow with perhaps one chromatography step to finish the purification.

http://www.pnas.org/content/97/9/4621.full

Here are comments on possible chromatography options:

3. The spin filters are too small; you need to concentrate the protein. The problem with the size exclusion step is that it will dilute the sample.

To concentrate the sample, you could use the anion exchange step as a first step. This will bind the protein and allow many contaminants to flow through. The lipopolysaccharide (LPS) has a high selectivity for anion exchangers, so your protein should elute in a lower concentration. With access to a chromatography system, you could equilibrate the anion exchange column in a low salt buffer with nonionic detegent, apply the sample, and elute with a NaCl gradient and collect fractions as described in the reference that I included in my last post. What dimensions are the Econo-Columns that you have available?

4. If you don't use centrifugation, you will need a second chromatography step to purify the protein. A single anion exchange step will never yield high enough purify. So you could use the conA for the second step. Another possibility for the second step would be to use hydroxyapatite, if that is available in your lab. Hydroxyapatite would bind your protein with a 5 mM phosphate buffer at ph 6.8 and could be eluted with a phosphate buffer gradient. I think hydroxyapatite would be a good choice because it would remove the LPS very efficiently. The LPS would elute at the very end of the gradient. Here's a link with more information on this product:

http://www.bio-rad.com/webroot/web/pdf/ ... n_2156.pdf

However, do plan to use the ConA if you have this available.

If you have access to chromatography system, there must be some columns or media available. Look again and let me know if there is anything at all. If you get good results on the first chromatography step and are able to concentrate the sample, you might be able to use the size exclusion step as the second step. But without the concentrator, you can't use the size exclusion step first, other wise your sample volume will be too high.

4. If you needed a third step (hopefully not), you could use the size exclusion step since you will have used two steps that concentrate the sample first. The tris-buffered saline or phosphate buffered saline would work well for this step.

5. You will use the SDS page gels to test samples to verify the presence of your protein in the flow through or the fractions. Have you run the SDS page gels? Do you have precast gels available to use, or do you have to make your own gels. Do you have standards to run that will work for a 38kD protein? Do you have another method available to verify the identify of the ompC during the purification? Do you have a mini gel module available? You can get the information with small gels; you don't need to run large gels.

6. Protease inhibitors are usually add to the cell lysate.

http://www.piercenet.com/browse.cfm?fld ... A875C0B771

Here's a lysis protocol that uses lysozyme and include PMFS as a protease inhibitor and includes a suggestion for an alternate from Roche. If you do use PMSF, be careful as it is toxic. It sounds like you don't have this item available.

http://web.mit.edu/king-lab/www/cookbook/plysis.htm

Look back at the article I posted and look at your other articles. I don't remember any protease inhibitors used in the chromatography steps. Some proteins are very sensitive to degradation and would require the inhibitors. If they are not included in that paper, then you can plan to work in the cold, and continue working once you get started. The minus 20 degree freezer is probably fine to use since it's for a short term storage. The sample should be frozen with glycerol, or you could add protease inhibitors if you have to stop the purification process for a few days. Once you lyze the cells, the minus 80 degree freezer is the best choice.

Let me know when you have more information available, or if there are any questions.

Donna Hardy