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| Question | Answer |
| costs associated with bringing recombinant protein to market | Research and development. Patenting. Scale-up. Culture. animal model trials. Clinical trials. Licensing. Production/advertising. Sales. Recoup costs and make profit (within patent). |
| what influences the choice of organism? | size of the protein product complexity of protein processing potential toxicity to host organism cost |
| plasmids can carry up to: | 6kb of foreign DNA |
| cosmids carry up to | 50kb of foreign dna |
| general organism issues (recombinant protein production) | 1.transcription termination by foreign gene sequence 2. codon usage may be problem 3. degradation of the foreign protein 4. protein produced may not work 5. may require post translation modification |
| advantages of e coli for recombinant protein production | 1.well studied 2. many vectors 3. grows quickly 4. produces large amts product 5. cheap |
| disadvantages of e coli for recombinant protein production | 1. human genes not expressed in e coli 2. transcription and translation signals differ 3. target gene must be surrounded with correct signalling codes 4. gene must be cDNA |
| somatostatin (first reombinant protein made in e coli- anti growth hormone) | 1.14 amino acids long 2. cloned gene into beta galactosidase gene of vector and producing a fusion protein 3. tx w/cyogen bromide to remove beta galactosidase |
| recombinant insulin (2 ways) | 1.make A and B chains seperately and form disulphide bonds chemically 2. make whole molecule and cleave C out with protease |
| if your protein is active as a single unprocessed polypeptide... | make it in e coli |
| If the activity of your protein is based on it’s complexity .... | Avoid e coli. Complexity can be co- and post-translational modifications that E.coli cannot or does not adequately perform |
| 7 post translational modifications of proteins | 1. glycosolation. 2 acetylation. 3 phosphorylation. 4. carboxylation. 5. methylation. 6. disulphide bridge formation. 7. proteolytic cleavage |
| Pros of yeast and fungi | eukaryotic. easy to transfect. cheap to grow. vectors for largest human genes. post translational modifications available |
| cons of yeast and fungi | do not use if correct glycosylation required |
| pros of insect cells | euraryotic. slow/expensive to grow. easy to trasnfect. vectors available. |
| cons of insect cells | glycosylation not identical to humans |
| plant recombinants | cheap. easy to produce. large amts product. not useful if protein must be modified. |
| mammalian cells for recombs | eukaryotic. slow growing/expensive. easy to transfect. vectors avail. active proteins. viruses are problem |
| glycosylation problems in higher eukaryotes | species dependent. humans and apes only species that do not have enzyme for Gal alpha 1-3 Gal |
| Gal alpha 1-3 Gal | immunogenic in man. found on many recombinant proteins and monoclonal antibodies |
| rationale for recombinant protein production | no batch to bath variation. safer. QC easier. |
| are recombinants really safer? | e coli prob virus free. if made in cells cultured with foetal calf serum-?BSE/CJD. horse products may have borna virus. any other animal-? viruses |
| all recombinants must be tested before human use | in vitro for determination of activity and purity. in vivo on animals. in vivo on humans. clinical trials |
| what are the in vivo animal tests looking at? | 1. dose. 2. response. adsorption. distribution. excretion. toxicity. |
| requirements for recombinant protien production | 1. gene must have been cloned 2. functional method avail to test product. 3. product must retain activity 4. product must be human compatible |
| Waht do we need to make a recombinant protien? | 1. gene encoding the protein of interest +/- promotors 2. vector specific to the organism we will use and big enough to carry gene 3.organism to be transfected 4.selection system, without which the non-transfected cells will outgrow those that have taken up the vector 5. a “tag” that will allow us to easily purify the product- one method used for every protein to be synthesised = economic sense 6. growth media, fermentors and downstream processing facilities |
| recombinant protiens | protiens sythesised by expression of a cloned gene in the cell of another species |
| where may recombinant protiens be produced? | number of different organisms: bacteria, yeasts, fungi, insect cells, mamallian cells |
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