Re: Possible Kickstarter projects

Hi John,

> Do you mean expensive to operate HPLC, or technically difficult to get
> results the same?

HPLC is both difficult to get/setup *and* difficult to do. It's not
something you could easily make a kit for and hand out; people need a
moderately advanced skillset to start playing with HPLC.

You could go with non-HP Liquid Chromatography..that is, tubes full of
adsorbent gels and resins etc., and possibly come up with a kit that's
more useful. But it wouldn't be very cheap to operate, I gather the
resins are very expensive per gram, and the procedure for isolation
would still be different for every enzyme. Hence my dislike of that
whole route.

> Sounds like a job for a kit manufacturer.

Kits for HPLC would be very welcome. I wouldn't have much use for one
(because I'd rather make them unnecessary for small-scale preps), but it
would beat the hell out of commercial offerings for those who want HPLC.

> What does elastin do?

Elastin-Like-Polymers (as opposed to Elastin itself) are a newish way of
purifying enzymes by translational fusion. In essence, you add a few
repeats of a simple amino acid sequence to the end of your protein, and
then express it in your cell lines. When you want to extract the
protein, you lyse the cells, add a little salt, and raise the
temperature to 37C. Filter/Centrifuge, cool, slice off the ELP, serve.

ELPs become insoluble at a temperature that depends on the ELP being
used, but once they hit this temperature they coagulate together,
dragging their conjugated protein out of solution while leaving all the
other cellular matter in solution. Once isolated, you can cool the
ELP-fusion protein and it will come back into solution again. I'm not
sure whether the ELP coagulation even denatures the main protein, so you
mightn't need to help it renature at all. If you want to use the enzyme
above 30C, you'll need to cleave off the ELP..so you use thrombin or the
like to cut the ELPs off. And Thrombin itself can be ELP purified. ;)

> So, does that mean you don't see a need to share load or buy
> from a distributor (and ship things
> around), just develop the right technique and it's DIY easy?

I think there's a market of course. After all, if the price is right I'd
rather not spend loads of time prepping up my own proteins. But, if I
can't afford commercial offerings I'd like to be able to make my own.
Also, in some countries you can't buy enzymes at all; even if they ship
to your country, your local currency might be worth nothing abroad, so
the already inflated prices are simply astronomical. I think the only
way to ensure everyone can get the enzymes they need is to make them
easily DIYable.

I suppose that means I'd never *need* to buy them, but I probably would
anyway.

--
www.indiebiotech.com
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