Obtaining Human Growth Hormone
An Independent Research Project Manipulating E. Coli Bacteria
The Human Growth Hormone (HGh) is a peptide hormone that stimulates growth, cell regeneration, and cell reproduction. It can be obtained using a bacterial expression system. This can be done by manipulating E. Coli bacteria to produce large amounts of human protein from the human gene . In other words, gene expression, or the production of proteins from genes, can be used to obtain these human proteins by using E. Coli bacteria.
The first step is to amplify the human growth hormone gene using PCR (polymerase chain reaction). We obtain one human chromosome containing two alleles for coding for human growth hormone. EcoRI and BamHI are two restriction enzymes used to make “cuts” on the alleles, so we are left with a EcoRI restriction site on one end and a BamHI Restriction site on the other end.The gene coding for HGh, which is between both restriction sites, is amplified using PCR. The next step is to digest the expression vector/plasmid, which is the circular DNA molecule of the bacterium with the restriction enzymes EcoRI and BamHI. The circular plasmid now has a small “gap” where there is the EcoRI site on one end and the BamHI site on the other end. These sites are DNA sequences. The plasmid also incorporates the AmpR gene, which is a gene coding for proteins that allow E.coli cells to resist the antibiotic ampicillin; it also serves as a selectable marker in bacteria. Next, the PCR product (the amplified HGh gene) is digested with EcoRI and BamHI. Then, ligation produces the new recombinant plasmid. This new recombinant plasmid consists of the PCR product ( the amplified human gene coding for Hgh) and the digested plasmid combined. The circular recombinant plasmid now contains the AmpR gene, the EcoRI site, and the BamHI site.
Now that the recombinant plasmid is formed, it needs to be introduced into the bacterial host cell via transformation, which is the process of introducing foreign DNA into a cell. The recombinant plasmid is put into the E. Coli host cell. It is then placed into a growth plate or Agar plate containing the ampicillin antibiotic. The ampicillin antibiotic in the growth plate only allows E. Coli cells that have the recombinant plasmids to grow.
Each bacterial colony that grows contains millions of E. Coli cells and each E. Coli cell produces millions of human growth hormone. Finally, the HGh is “purified” by breaking apart the plasma/ cell membrane of E. Coli bacteria to harvest human growth hormone protein. This results in 99% pure human growth hormone protein.