How can I produce large amounts of recombinant proteins in a relatively simple manner?

Question

I have a science project. The project is to prove that insulin and other protein/peptide hormones can be synthesized in a fairly simple and inexpensive manner, using machines and materials that a slightly above-average laboratory would have, or would have the funds to buy.

I need more information (including very specific laboratory protocols) on how recombinant proteins ARE synthesized, as well as how they CAN BE theoretically synthesized. Keep in mind that keeping cost of materials down is a priority second only to producing the proteins.

Please do not reply saying I should have chosen an easier project.

Answer 1

Most recombinant proteins in laboratories are produced using bacterial expressions systems, typically the species is E. coli. In short, the gene that codes for that protein is inserted into a piece of circular DNA called a plasmid. Plasmids contain a gene for resistance to an antibiotic. One of the easiest ways to purify your protein is if you combine it with a small protein tag that is used solely for purifying it. Very common is what is called a "His tag". This is just 6 or so histidine amino acids in a row at one end of your protein. Several plasmids designed for protein purification have His tags built in, all you have to do is get your DNA in the plasmid in frame with the codons for the tag.

Insert the DNA into the bacteria through a process called transformation. A very simple method of transformation (called "Heat Shock"): Put the bacteria in a tube on ice. Add DNA, seal the tube and wait 30'. After 30', put the tube (still sealed) in a water bath that is 42 degress centigrade for 45 seconds. Then, back on ice for 2 minutes. Then, add some bacterial grown medium and put the tube at 37 degrees for about an hour. After this, you spread the bacteria on agar plates (also made with bacterial media) that contain the same antibiotic as the resistance gene on the plasmid and leave it at 37 degrees overnight. The following morning, the colonies of bacteria on the plate will contain the plasmid with the gene you want to express.

From this point on, as long as you keep the antibiotic around, the only bacteria that grow will contain the gene for the protein you want to express.

Next: pick a colony of bacteria and transfer it into liquid media (still with antibiotic), and grow it at 37 with shaking until the bacteria reach "mid-log phase." Note, E. coli growth has a doubling time of about 30 minutes. Depending on how much you grow, this step may take several hours. Mid-log phase is when the bacteria are growing very rapidly and still reasonably dense, but not overgrown. At this point, you induce expression of the protein by adding a chemical to your soup. The most common chemical is IPTG, but it is specific to the plasmid.

Now, keep your bacterial at 37 in the liquid media, and shaking for 1-3 hours (depends on the protein to be made).

Stop the growth, and spin down your bacteria in a cold centrifuge (4 degrees centigrade). Bacteria will settle without spinning them, but you want them to stick to the bottles so that you can pour away the bacterial growth media.

Resuspend the bacteria in a simple buffered salt solution (precise details of the solution can vary depending on the protein you want to get out). Break open the cells. Lysozyme and detergent can be used, as they are cheap. Then, spin out the broken cells in a centrifuge so that only the stuff released when the cells are broken is present.

Isolating protein: If you have a His-tagged protein, you can use a nickel matrix, or cobalt matrix to purify your particular protein. Histidine resides will bind to charged nickel or cobalt and when you have 6 of them in a row on your protein, they will bind to these matrices (essentially very small beads) selectively. Collect the beads and get rid of the rest. REmember, to never let your beads or protein dry out, they must remain moist or wet at all times. Wash the beads to remove anything non-specific if you are concerned about purity.

Note: These nickel and cobalt matrices are probably the most expensive part of the procedure, but can be washed, stripped and re-generated so that they can be reused again at a later date.

You can get your protein off the beads by incubating them with a chemical called imidazole. Imidazole is much like having free histidine in solution. It competes with the histidine in your protein to bind to the nickel or cobalt and as a result, if you have enough of it, your protein unbinds and is now free in solution.

Plenty of steps can be added to make it more complicated, to increare your yields, and maybe some others have shortcuts, but this is the essence of what I have done for several years in my own laboratory.

There are also expression systems that rely on yeast to produce the protein. The most common laboratory yeast is Saccharomyces cerevisiae, but Pichia pastoris is now being routinely for production of eukarytic proteins.

Also, scientists can get proteins from milk (it is secreted into the milk), and even from plants. But bacteria are the most common way to make recombinant proteins in laboratories. They might not be the favored method when the proteins or peptides are intended as drugs or for humans, but they are incredibly common for research purposes.

Answer 2

Depends on the scale you want to produce. For super small you need:
1. The gene of interest spliced into your host organism.
2. A shaking incubator to begin a starting culture for say 250mL.
4. A mechanism to burst or lyse the cells.
5. Small centrifuge to seperate cell debris.

Bingo, you got some rough protein soup. Purify if necessary. Assay/Analyze to check out what you got! Its fairly cheap too.

Answer 3

The way this typically works is that the entire gene for the protein is cloned into a bacterium or yeast that can, in turn be grown in large volumes. The protein gets expressed in the bacteria, which can be harvested and the proteins extracted by standard biochemical techniques.

Answer 4

The answer Jerry P gives is good. The process is cheap, simple, effective.

Answer 5

masturbate at least once per day and this will of course at least double the amount of recombiant proteins you produce over time and it will also get your mind off such geeky Q's for a while each day and you might actually get around to living life a little

WIN WIN !!!

Answer 6

You could just write "I believe in evolution" and then "This had to happen naturally at some point". If your teacher/professor is a hard-nosed evolutionist, he won't question his own belief system.