Enzyme-resistant Biotin Linkers

[DanBayley]

Hey, I just came across this technology that involves a biotin-based biological binding method with increased enzyme resistance for in vivo targeting and more. I thought this was pretty awesome because it can provide non-radioactive targeting (for specific compounds).

Does anyone know if that would be possible with this technique? Or maybe it's better suited to something else?

You can see the details here: http://marblar.com/challenge/biotin

[Knumbnuts]

Well you need to link the biotin to your specific compound via a spacer. Then react it with your enzyme or protein or whatever. Then you put the bound substrate down a streptavidine coated column. This binds the biotin end of the substrate/enzyme/receptor complex.

Once you have identified the column fractions containing the material of interest you displace the complex by treatment with excess biotin.

This takes a lot of work, getting the spear length correct, making sure this bitionylated compound binds properly, properly functioning assays for your target of interest and so on.

But when it works it is not a bad method.

[Horza2002]

Yes it will work because I have done this in our lab. We have done it on a number of enzymes and a number of natural product analogues to aid in their purification. As in the link, a click reaction is usually used to introduce the biotin linker to the substrate/enzyme of interest as it is very easy to do.

 

There are several limitations to this procedure though. Binding of the biotinaylted substrate is not always easy. We found that the larger the substrate you try to trap, the harder it is to bind. In addition to this, you need to use alot of biotin to elute your trapped substrate from the strepavidine column. Commerical biotin is really really expensive at the moment and so doing this on a large scale is not really practical. It is possible to overproduce biotin from microorganism and purify it, but this is a very lengthy and time consuming process.