Transformation (protocol: attachment 2)
The five constructs mentioned in the materials have to be transformed into different E. coli strains. This is done by heat shock at 42˚C. Meer info
Periplasmic extraction (protocol: attachment 3)
Expressed Proteins go via the Sec pathway to the periplasmic space. This is a first separation step of proteins. With periplasmic extraction the outer membrane of an E. coli cell will be ruptured by an osmotic shock caused by sucrose releasing all the periplasmic proteins. Cell lysis (protocol: attachment 5) will rupture the cell, but also the inner membrane causes to release all cell components (proteins, organelles etc.). Then it could be very difficult to separate folded and non-folded proteins properly. The (TES) buffer containing sucrose (20%) also contains Tris, EDTA and Protein inhibitor. To respectively control the pH, bind free metal ions and to diminish protease activity. After releasing the protein centrifugation at 4˚C will separate the cells and proteins. An additional osmotic shock is done by TES buffer with MgSO4 (salt) to release the last of the protein residing in the periplasm. After centrifugation at 4˚C the supernatant contains all the periplasmic proteins. Centrifuge steps to pellet the cell culture is done by Rc-5 Superspeed Refrigerated centrifuge of …show more content…
When the salt concentration is increased, water molecules interacts with salts in solution, which decreases the number of water molecules available to interact with the charged part of the protein. As a result the protein-protein interactions are stronger than the solvent-solute interactions; the protein molecules coagulate by forming hydrophobic interactions with each other and
The five constructs mentioned in the materials have to be transformed into different E. coli strains. This is done by heat shock at 42˚C. Meer info
Periplasmic extraction (protocol: attachment 3)
Expressed Proteins go via the Sec pathway to the periplasmic space. This is a first separation step of proteins. With periplasmic extraction the outer membrane of an E. coli cell will be ruptured by an osmotic shock caused by sucrose releasing all the periplasmic proteins. Cell lysis (protocol: attachment 5) will rupture the cell, but also the inner membrane causes to release all cell components (proteins, organelles etc.). Then it could be very difficult to separate folded and non-folded proteins properly. The (TES) buffer containing sucrose (20%) also contains Tris, EDTA and Protein inhibitor. To respectively control the pH, bind free metal ions and to diminish protease activity. After releasing the protein centrifugation at 4˚C will separate the cells and proteins. An additional osmotic shock is done by TES buffer with MgSO4 (salt) to release the last of the protein residing in the periplasm. After centrifugation at 4˚C the supernatant contains all the periplasmic proteins. Centrifuge steps to pellet the cell culture is done by Rc-5 Superspeed Refrigerated centrifuge of …show more content…
When the salt concentration is increased, water molecules interacts with salts in solution, which decreases the number of water molecules available to interact with the charged part of the protein. As a result the protein-protein interactions are stronger than the solvent-solute interactions; the protein molecules coagulate by forming hydrophobic interactions with each other and