Sample Description

Dear collaborator

In order to have as much information as possible about your sample before planning the experiments we ask you to kindly fill in the Service package order forms. The information you supply will increase the chance of success. Your sample is expensive and precious, and this information will also reduce the risk of wasting sample on trials that in retrospect should have been avoided. In addition we have prepared a set of guidelines which may be helpful for you to prepare your sample.

SP1 Protein production order form
SP2 Structure determination order form
SP3 Drug discovery and design order form

SP1 Protein production guidelines
SP2 Structure determination guidelines


SP1 Protein production guidelines

Introductory remark
If the aim of the protein production is structural analysis using protein crystallography, we recommend people to make gene constructs with a fusion tag that be cleaved off. Although tags help immensely in the purification steps, most tags are prone to cause problems in the crystallization process, especially if the linker region between the protein and the tag is long and thereby floppy. We recommend introducing a cleavage site for TEV protease between the tag and the protein.

Cloning and small scale expression and purification
The standard package (SP1.1a) includes cloning of a gene into an E. coli expression vector using restriction enzyme independent cloning (Invitrogen). The gene should be supplied in a form such that it easily can be inserted into the vectors we use for expression, preferably as genomic DNA or cDNA. Vectors already prepared for the Gateway system should be avoided because the gene cannot be excised from the vector and inserted into a new plasmid if required.

Medium and large scale expression and purification
The gene (with N- or C-terminal His-tag) should be supplied transformed into the cell line of the customer’s choice, preferably as a cell culture excised from an agarose plate or a 1 mL cell culture. Confirmation of small scale expression in this strain has to be supplied, eg. as visualized on an SDS gel, before NorStruct starts medium and/or large scale expression. Confirmation of a small scale purification procedure providing a purity adequate for the customers purpose, has to be provided before medium and large scale expression is started. Included in the packages is purification using His Trap columns and gelfiltration. In the event this is not sufficient for the purity required, special customization may be agreed upon. NorStruct cannot guarantee that scale-up will provide the exact same expression levels as the customer may have seen in small scale expression.


SP2 Structure determination guidelines

For setup of crystallization experiments we do have a set of guidelines, but these are by no means very strict. Proteins behave differently and this means that they have to be treated individually. The guidelines listed below are however applicable to most of the proteins we are dealing with.


In order to perform an initial broad and efficient screening for crystallization conditions we require about 3 mg of protein. Of course, more is always better. We prefer to receive the protein in a solution at 10 mg/ml. If the protein is very soluble the concentration can be higher, and similarly, if it is poorly soluble, the concentration could be less. However, our experience is that concentrations less than 1 – 2 mg/ml can be very difficult to crystallize. If solubility is less than 1 – 2 mg/ml one should try to add various additives, e.g salt or detergents, to see if the solubility can be increased.

The protein could be stored in most buffers used in expression and purification, but phosphate buffers should be avoided. This is because phosphate precipitates with most metals used in crystallization solutions. Phosphate buffers should only be used if no other buffer could be used. It should be noted that particularly Tris and BisTris are very temperature sensitive and the pH can change a lot.

We prefer to store the protein in several tubes with small volumes, and not all in one tube. This way we don’t have to thaw all of the protein when we perform the experiment, since most proteins don’t like to be frozen and thawed several times. We generally use only a small volume of the sample each time. We generally store the protein frozen (if the protein can be frozen) aliquots of e.g 50 µl. Even if the protein doesn’t need to be frozen it can be a good idea to keep it in small volumes in case of contamination. We discourage people from freeze-drying the protein.

Ideally the protein should be 100% pure, but above 95% purity is more realistic. How pure a protein should be will also depend on how much you loose when trying to purify it. It’s a compromise which often only can be found by trial and error: Some proteins HAVE to be absolutely pure in order to crystallize whereas others don’t care.

We tend to stress that when running an SDS gel in order to estimate purity you have to make sure that you apply A LOT of sample. If the sample is too dilute one will not see the impurities. We generally want to see a picture of the gel before setting up crystallization experiments.

We generally encourage people to always include a gel-filtration step, but of course, if you loose all your protein in this step, you don’t do that.

If your protein is produced with a tag it can always be debated whether it should be removed or not prior to crystallization. If the linker region between the tag and your protein is long, this is almost doomed to cause problems in crystallization, so most people cleave off such a tag. If the linker, on the other hand, is only a few residues, the protein may crystallize successfully.


 



Ansvarlig for siden: Ronny Helland
Sist oppdatert: 11.05.2011 00:47

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