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Recombinant protein and its expression system - Coggle Diagram
Recombinant protein and its expression system
what is recombinant protein?
manipulates form of protein, which generated in various ways to produce large quantities of proteins, modify gene sequence and manufacture useful commercial products
Detailed study of protein expression
DNA techniques, RNA techniques, protein expression techniques, protein purification and production
Large scale protein production
Clinical relevant proteins
Eg, insulin, interferon s, IL-2, Somatotropin, Erythopoietin
Pros
pathway engineering is
very specific
for easy manipulation depending on host cell and protein desired
Greater copy number of genes
in higher quantity of product
can use high-copy plasmid
prevent plasmid
lost
by genome intergration of DNA
Cons
large scale production and purification is
extremely difficult and precise
high-copy plasmid may be
unstable/redundancy
may occur
can be
difficult to integrate
multiple copies of gene into host genome due to unreliability of multiple gene copy intergration
Process
Determining DNA,RNA,Protein sequence
Sequence techniques
PCR and RT-PCR
gDNA and cDNA libraries
Cloning of correct gene into expression vector for enhance production
Restriction endonuclease digestion
Gene intregation and ligation into vector
Transformation of vector into host cell and expression
cold shock and Ca treatment for transformation
Gene intregation into gDNA
heat shock
Prokaryotic vs Eukaryotic cell use in protein expression
Prokaryotic cells
Easiest cells to grow and genetically manipulated
Antibiotic resistance genes for increased selectivity for transformed bacteria
lack before and after translation protein modification pathways for correct protein manufacturing
Eukaryotic cells
not all genes are able to be expressed in prokaryotic cells
has all necessary promoters and terminators in gDNA already
Expression system
Bacteria expression system
Advantage
clear genetic background
fast breeding
low cost
high expression level
easy purification
good stability
strong anti-pollution ability
wide application range
Disadvantage
hard to purified inclusion bodies
imperfect post-translational modification processing system
low biological activity
Yeast expression system
Pros
similar to bacterial protein expression
naturally occuring plasmid
secretes few proteins for easy purification of recombinant protein
able to carry out many post-translational modification
Cons
not suitable for high-density culture
lacking strong and strict regulation promoter
low secretion effciency
baculovirus expression system
Cons
culture medium
oxygen supply
logarithmic growth
Pros
recombinant protein has complete biological function
post-translation modification
high level of expression, up to 50% of the total protein
accommodate large insert protein
simultaneously express multiple genes
safer to use than mammalian virus
Mammalian expression system
Transient expression system
short protein expression duration
simple
short time
Stable cell line generation
time consuming and laborious
protein expression last a long time
Pros
initiation signals
protein processing
protein secretion
protein glycosylation
intact macromolecules
native structure
Cons
complicated
high requirement
low yield
sometimes viral infections
pET vector expression system
Consist of 4 sites
site of transcription with lac operon and gene of interest
origin of replication and antibiotic resistance gene
Lac I for production of Lac operon repressor protein
Normal function - No protein expression
Lac I protein represses transcription by preventing T7 RNA polymerase expression
Altered function - Protein expression
IPTG is added to induce protein expression
IPTG binds to Lac repressor protein and expresses T7 RNA polymerase for transcription