Question | Answer |
Blotting used in making a recombinant protein | Southern Blotting |
Best host cell for small simple proteins | bacteria and viruses |
complex, glycosalated proteins would need what kind of cell | mammalian cells |
batch culture | a few generations grow
environment conditions get used up |
fed batch culture | growth limiting nutrient |
continuous culture and production | maintained under constant conditions with a constant nutrient supply
the microorganism can grow steadily for a long period of time |
stirred tank bioreactor | most reliable
stirred to generate mixing for mass transfer
stirred with paddles |
airlift bioreactor | stirred with air flow instead of paddles |
microcarrier bioreactor | fixed bed biorectors
support matrix that allows for cells to grow
carrier material may be: gelatin,collagen or cellulose |
membrane bioreactor | hollow fiber perfusion
uses membrane and bioreactor to cultivate and purify |
Mediums that are used for cell culture | fetal calf/synthetic serum, vitamins, amino acids, carbon source, water, electrolytes (Ca, NA, K, PO4), trace minerals (Fe, Mg, Cu, Zn, etc.), and hormones (growth factor) |
contaminates | viruses, bacteria, cellular DNA, protein |
precautions | wear latex gloves, use sterilized glassware instead of baked, use treated water, clean all utensils |
cdeal with contaminants by: | centrifugation, filtration, selective precipation and chromatography |
scale-up: | number of processes used to isolate a certain protein
produce a product at a high quality at a competitive price |
Step1 of scale up: | particulate removal- removal of cell debris |
step 2 of scale up: | concentration-take everything that was not removed and put it back together |
step 3 of scale up: | protein capture-isolate the protein that you need (locate it) |
step 4 of scale up: | removal of contaminants (final purification)-remove any contaminants |
step 5 of scale up: | sterilization-finalize the product, prepare drug for the patient |
size-exclusion chromatography | separates proteins based on their size |
reverse phase chromatography or hydrophobic interaction chromatography | separates the proteins based upon the hydrophobic properties of some proteins |
ion exchange chromatography | separates proteins based on their charges |
affinity chromatography | how well a protein binds to a ligand |
transgene | foreign gene placed in the embryo and the gene develops as the animal matures |
pharm animal (transgenic) | animal that the transgene is inserted into
it expresses the gene |
inclusion bodies | nuclear or cytoplasmic aggregates of sustainable substances, usually proteins |
why can inclusion bodies be problematic? | A lot of you product is being tied up in the inclusion bodies so you do not get the yield that you want |