Know Your Protein

by | May 6, 2019

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As SPR is generally used for assessing binding to proteins in one form or another it’s a good idea to ensure that you have as much information about the proteins you’re assessing as possible…

This isn’t only important to know in order to save time and money, but because a poor understanding of your protein can lead to poor and often inaccurate data. Here we discuss the importance of knowing the protein’s primary sequence, what effect this has on the isolectric point (why you should care), and also what you should be looking for when purchasing protein. The goal is to arm yourself with the best information to make the best start possible.

In the beginning

Where do you start? Generally, in the CRO/CTO world the client will either supply or say what protein(s) they want to investigate but if that’s not your arena then scouring the literature is a great way to get off the mark. Once you have a protein in mind, it’s vitally important that you know the primary amino acid sequence as this is what ultimately determines its final degree of structure and the isoelectric point.

The isoelectric point is the pH at which the protein carries a net charge of zero. Knowing this will help you pinpoint the sodium acetate buffer that you would initially choose to immobilise your protein.

With regard to primary sequence, it’s also important to know that you have the same sequence that other people have used in order to ensure that you’re measuring the same interaction. CD20 is a great example here as most protein suppliers only express the internal region (residues 213 – 297) and not the external loop region (residues 141 – 188) that binds an antibody such as Rituxan. So, it’s definitely wise to look at the residue numbers!

Buying good protein.

At some point you’re going to have to buy proteins that don’t come in a kit and at this point it’s definitely caveat emptor! There are many excellent suppliers out there, but here are a few things I look for when it comes to trying to source the best possible protein:

Purity

Ideally you want the protein to be >95% pure by SDS-Page and contain no carrier protein (BSA) or trehalose. BSA can be a pain for SPR if you’re trying to immobilise a protein, and although it can help issues with non-specific binding, it can also cause more hassle than it’s worth. If you do have to have BSA in the protein, make sure BSA is also in the running buffer. Trehalose will becovered in much more detail in a future post, but in short it adds to the refractive properties of the assay if the protein is used as the analyte.

Source

Seems obvious, but it’s important to make sure that your protein sequence comes from the correct species otherwise you run the risk of assessing the wrong interaction. A quick search of VEGF results in several different species I can choose from, so make sure you choose the correct one and that it contains the correct primary amino acid sequence and predicted N-terminus for your needs.

Expression System

There’s very little point making conclusions from proteins that aren’t expressed in a cell that can fold and post-translationally modify proteins like you’d expect to find them in nature. For example, the Fc receptors are heavily glycosylated so you wouldn’t want to use an expression system such as E.Coli to make them as the results won’t reflect what’s happening in nature. Therefore, HEK293, CHO and insect cells are a safe bet for most proteins.

Tags

This is a huge area and can and will have a massive impact on your assays. It’s worth considering that purification tags can and do have an impact on the protein’s tertiary structure, affecting its function if it’s an enzyme or binding if it’s an antigen. Even a small tag such as Hexahistidine (~ 1 kDa) can cause changes so it’s always best to choose the tag that is most suited to your needs and to ensure that the tag location does not interfere with the protein’s function. My personal preference for the ‘best’ tag is biotin but only when it’s done properly using an avitag.

His tag
Probably the most common purification tag on the market with several good anti-histidine antibodies capable of detecting mixed His tags (non-selective), allowing the use of one antibody for all assays. The main benefit of His tag capture is that the surface can be easily regenerated using a low pH Glycine solution and the antibodies, in general, can handle multiple regenerations before any decrease in capture efficiency or baseline drift is noted. As a rule of thumb, the strength of capture is directly linked to how many histidines are in the tag so a deca-tag is often better than a hexa-tag protein.

Biotin-streptavidin
Personally my favourite tag. The main advantage of Biotin-streptavidin is that biotin is small and stable, and labelling of molecules rarely interferes with the function, making it a good choice for capture. Given the increased frequency of avitag and BirA usage, it is now feasible to produce site specific biotinylated molecules in large quantities.

Fc tags
Fc tags are my least favourite protein purification tags even though they allow assay specific orientation. Obviously if you’re measuring an analyte without an Fc region then the Fc tagged proteins become very useful.

Example

Vascular endothelial growth factor VEGF is a good example of a protein where there’s many different isoforms and as result many small differences in the primary sequence. For VEGF-A there are at least eight isoforms, each with different exons expressed and ultimately a different function.

Vascular endothelial growth factor VEGF is a good example of a protein where there's many different isoforms.

When we align the isoforms we see small differences in the general sequence until we get closer to the C-terminus which will affect your isoelectric point. With the simple inclusion of exon 6a, we notice that isoforms 189 and 206 have massive changes in charge at pH 7.4 (and in general across all pH ranges). For the pH values I’ve used Sodium Acetate which are commercially available from GE Healthcare at pH 4.0 – 5.5.

VEGF Variant vs Isoelectric Point

Summary

There you have it, a short overview of how to better understand the effect your protein’s structure on binding, and on what to bear in mind when it comes to sourcing those proteins. With this, we are ready to look at materials and buffers…

Would love to hear your thoughts on challenges you have had with primary structure, the ‘good, the bad and the ugly’ of protein sourcing and if you have any specific questions on your own assay set ups…

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