recombinant protiens

Description

Molecular Theraputics (molecular theraputics) Flashcards on recombinant protiens, created by lumen7 on 24/04/2013.
lumen7
Flashcards by lumen7, updated more than 1 year ago
lumen7
Created by lumen7 over 11 years ago
64
1

Resource summary

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
Show full summary Hide full summary

Similar

transgenic animals
lumen7
Stem Cells
lumen7
Gene silencing
lumen7
simple protein therapy
lumen7
Theraputic antibodies
lumen7
NICE/Helsinki
lumen7
Section A questions
lumen7
Molecular Theraputics
lumen7
vaccines
lumen7
Cancer Gene therapy
lumen7
Transplantation
lumen7