What is the procedure to clone Human Growth Hormone?

Question by Think: What is the procedure to clone Human Growth Hormone?
Whether it is by protein secretion technology or Recombinant DNA technology or mouse-cell technology, I need to know the exact method stepwise, the vectors/phages used, with diagrams if possible.

If anyone can explain it to me or if u could direct me to some weblink, it would be highly appreciated. (Wiipedia is not of much help here).

Thanks.


Best answer:

Answer by Chris W
You're right, the information under Wikipedia's Growth Hormone Treatment section is very vague. However, it does tell you that the gene for the growth hormone is inserted into bacteria, which mass produce the protein.

From that, we can surmise what you could potentially do to mass produce growth hormone.

First, of all, you need the gene. That's simple. You get a bunch of pituitary cells and then create some primers that bind upstream and downstream of the actual gene.

These primers should consist of approximately 25 base nucleotides, and should bind before the gene and after the stop codon.

You then use PCR to amplify it up. That technique can be found in any old Molecular Biology textbook, though the Maniatis book (referenced below) is very good and detailed on this.

Now the best thing to do next is to put your gene of choice into a plasmid (a circular piece of DNA) that expresses an antibiotic resistance gene, and right in front of a gene promoter that is always switched on (and obviously, one that forces the bacteria to produce vast quantities of the hormone).

That's very simple. Use a nuclease to cut your plasmid. (It's best to use two nucleases, to cut out a small section of the plasmid DNA out). Use the same nucleases to cut the ends of your extract gene (making sure the nuclease that cuts upstream of your extract DNA is also the same nuclease that cuts at the "upstream" part of your plasmid and vice versa). That means you have to find a nuclease that cuts at the right places, which may require a bit of searching.

Because both have been cut by the same enzyme, they'll have sticky ends that match. They'll anneal naturally and because you used the same enzyme to cut upstream of both plasmid and extract gene, the gene will anneal the right way round instead of backwards. (If it anneals backwards, the gene won't work). Use a DNA ligase to join up the backbone and voila, you've got a plasmid that should express antibiotic resistance and your hormone gene.

Transform it into bacteria, possibly using heat shock. Select for the bacteria that express the antibiotic gene (as it should also express the growth hormone), by putting it on to an agar plate with the antibiotic in and lots of goodies and nutrients for the bacteria.

Only the ones with the plasmid will survive and they'll form little colonies on a plate. Now you've got to check that your plasmid contains the gene. After all, the insertion of your gene into the plasmid could have failed.

That's simple. All you have to do is take a little bit from a colony, break it open and use a pair of primers that bind at the 5' end and the 3' end of the gene, and do a PCR. If the gene is there, you'll get a product. If not, you won't.

Then, you do a Western blot, to make sure the protein is actually being made. You could have skipped the above PCR step, but there was the chance that some rogue growth hormone could have infected your samples. Doing the PCR, shows you that any protein is there because the bacteria is making it from the gene you put into it, and not because it just happened to be on some of your apparatus.

I'll refer you to the sources below, as a Western blot is a long detailed procedure that'll make this answer insanely long.

After you've done that and proved to yourself that the bacteria has your gene and is making your protein, you grow it up in a huge vat full of nutrients. Siphon some off every now and then, and extract the protein.

You need do nothing more, except keep growing the bacteria, making sure it's always there and that the antibiotic selection is always on. If it isn't, the bacteria will eventually reject the plasmid, as it won't be of any use to it.

There you have it. It may not be the exact same protocol as what industry uses, but it will get you similar results.

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