gorilla_boy said:
If you actually know what you're talking about then please describe to me (in layman's terms if possible) the process for removing GH from a pituitary gland and whatever equipment it takes to make it usable contrasting it against the process and equipment required to synthesize rHGH.
I'm betting that taking it from the pituitary gland is easier.
Gorilla_boy, I do this sort of shit for a living. I know what I'm talking about. Don't call me a defensive asshole when you're the one taking offence at a small, calm statement. Bullshit like this human pituitary extraction you're promoting is how rumours get started and live on as urban legends you tell your kids: "don't use GH - it's from dead bodies' pituitaries!"
From the pituitary:
-obtain cadaver tissue and remove relevant gland.
-homogenize tissue and prepare crude soluble extract (hard spin to pellet extraneous DNA, cell membrane, organelles, etc.)
-from soluble extract, perform crude salting out (ammonium sulfate cuts) and localize fraction which contains maximal hGH concentration. Lyophilize (freeze-dry) to minimize volume if desired.
From this point on you can take a number of steps, but the most likely ones would be applying a rehydrated (smaller volume) lyophilized sample to some sort of affinity column for a rough purification. Failing that, large-scale preparative HPLC could be run - the equipment for this alone would total millions for a commercial operation. There are many methods for preparation, and the process would almost certainly require a number of steps. You're trying to remove a single, low-concentration protein in MILLIGRAM amounts (for protein work, this is a TON) from a proteome of hundreds of thousands of proteins and millions of degradation products and other contaminating chemicals found within mammalian cells.
You're looking at a recovery of maybe 100ug of hGH from each pituitary. MAYBE. So for a 100iu (about 33mg) kit, you're looking at a lot of stiffs... not really cost effective.
From a recombinant
E.coli strain:
-put the gene for hGH behind a promoter which is easily inducible. Quite often this is the lacZ promoter which is easily manipulated and is activated very strongly in the presence of lactose.
-transform
E.coli with this plasmid, or integrate the gene construct (i.e. lacZ:hGH). It would be preferable to use a high copy-number plasmid. This sort of transformation could be also done with a baculovirus expression system over insect cells, or in yeast.
E. coli would be the easiest to work with as far as total proteome, however, as many strains exist with a minimalized proteome of only perhaps 1,000-10,000 proteins instead of hundreds of thousands in mammalian cells.
-Grow the cells and induce the promoter (add lactose to the growth medium). This will stimulate mega-levels of expression of your target protein.
-Lyse the cells gently (i.e. by sonication or by addition of lysozyme). NOW, if your protein was tagged with an immune epitope (e.g. myc, HA, etc. can be added to the protein via genetic constructs, and they won't necessarily affect the function of the protein) you can run this over an immunoaffinity column - still less expensive than the equipment for large-scale prep HPLC, but maybe hundreds of thousands instead of millions for set-up costs - and have highly pure bound GH in a single step.
-wash away contaminating proteins with low-salt elution buffers.
-elute your hGH in high salt elution buffer, lyophilize the eluate.
-re-dilute, assay for protein purity and content, aliquot into vials, lyophilize, seal and distribute with vials of appropriate diluent.
Ferment 10,000 L of culture and you might get yourself kilograms of hGH in a single pass (with a large enough column). THAT is cost effectiveness.
-M