Flashcards
recombinant protiens
Flashcards do conjunto (28)
- 1costs associated with bringing recombinant protein to marketResearch and development. Patenting.
Scale-up.
Culture. animal model trials.
Clinical trials.
Licensing.
Production/advertising.
Sales.
Recoup costs and make profit (within patent). - 2what influences the choice of organism?size of the protein product
complexity of protein processing
potential toxicity to host organism
cost - 3plasmids can carry up to:6kb of foreign DNA
- 4cosmids carry up to50kb of foreign dna
- 5general 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 - 6advantages of e coli for recombinant protein production1.well studied
2. many vectors
3. grows quickly
4. produces large amts product
5. cheap - 7disadvantages of e coli for recombinant protein production1. 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 - 8somatostatin (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 - 9recombinant 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 - 10if your protein is active as a single unprocessed polypeptide...make it in e coli
- 11If 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
- 127 post translational modifications of proteins1. glycosolation. 2 acetylation. 3 phosphorylation. 4. carboxylation. 5. methylation. 6. disulphide bridge formation. 7. proteolytic cleavage
- 13Pros of yeast and fungieukaryotic. easy to transfect. cheap to grow. vectors for largest human genes. post translational modifications available
- 14cons of yeast and fungido not use if correct glycosylation required
- 15pros of insect cellseuraryotic. slow/expensive to grow. easy to trasnfect. vectors available.
- 16cons of insect cellsglycosylation not identical to humans
- 17plant recombinantscheap. easy to produce. large amts product. not useful if protein must be modified.
- 18mammalian cells for recombseukaryotic. slow growing/expensive. easy to transfect. vectors avail. active proteins. viruses are problem
- 19glycosylation problems in higher eukaryotesspecies dependent. humans and apes only species that do not have enzyme for Gal alpha 1-3 Gal
- 20Gal alpha 1-3 Galimmunogenic in man. found on many recombinant proteins and monoclonal antibodies
- 21rationale for recombinant protein productionno batch to bath variation. safer. QC easier.
- 22are 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
- 23all recombinants must be tested before human usein vitro for determination of activity and purity. in vivo on animals. in vivo on humans. clinical trials
- 24what are the in vivo animal tests looking at?1. dose. 2. response. adsorption. distribution. excretion. toxicity.
- 25requirements for recombinant protien production1. gene must have been cloned 2. functional method avail to test product. 3. product must retain activity 4. product must be human compatible
- 26Waht 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 - 27recombinant protiensprotiens sythesised by expression of a cloned gene in the cell of another species
- 28where may recombinant protiens be produced?number of different organisms: bacteria, yeasts, fungi, insect cells, mamallian cells
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- Molecular Theraputics (molecular theraputics) FlashCards sobre recombinant protiens, criado por lumen7 em 24-04-2013.
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