recombinant protein and its expression systems
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Recombinant Protein and Its Expression Systems
What is Recombinant Protein ?
What does Proteins do?
This is what we have in nature
This is the quantity and purity we want
Why We Need Recombinant Protein
“Recombinant protein is a manipulated form of protein, which is generated in various ways to produce large quantities of proteins, modify gene sequences and manufacture useful commercial products.
Recombinant protein is encoded by recombinant DNA, which has been cloned in a system that supports expression of the gene and translation of mRNA.
Ori
pCDNA3.1
5428bp
Amp r
Neomycin
SV40 ori
PCMV
f1 ori
BGHpA
SV40 pA
T7
Recombinant DNA/ Vector
Host Cell+ Vector
Host Cell Proliferation
High-level Protein Expression
Recombinant Protein Technology
Choose Suitable Expression System
Target Protein PropertyApplicationProtein YieldCostPost-translational Modifications…
Choose Suitable Expression System
Bacteria Expression System
Yeast Expression System
Insect Expression System
Mammalian Expression System
Bacteria Expression System
Advantage:
Clear genetic backgroundFast breedingLow costHigh expression level
Easy purificationGood stabilityStrong anti-pollution abilityWide application range
Bacteria Expression System
Disadvantage:Hard to purified inclusion bodies
Inclusion Bodies
Aggregation
Bacteria Expression System
Disadvantage:
Imperfect post-translational modification processing system
Low biological activity
Yeast Expression System
Sequenced in 1996 Used in brewing and bread industry for thousands of years
Yeast Expression System
Disadvantage of S.cerevisiae :
Not suitable for high-density culture
Lacking strong and strict regulation promoter
Low secretion efficiency
Yeast Expression System
Methanol Yeast System Host Cells:
H Polymorpha
Candida Bodini
Pichia Pastoris
Fission Yeast Methanol Yeast
Yeast Expression System
Yeast Expression System
Nucleus
Cytoplasm
Mxr1
Mxr1
Glucose
Methanol Prm1 Mit1 Aox1
Advantage:Production up to gramsTranslation closer to mammalian cellsDon’t undergo hyperglycosylation
Baculovirus Expression System
Generation of Recombinant Baculovirusand Gene Expression
Limitation Factors:
Culture medium
Oxygen supply
Logarithmic growth
…
Baculovirus Expression System
Baculovirus Expression System
Advantages:
Recombinant protein has complete biological function
Post-translation modification
High level of expression, up to 50% of the total protein amount
Accommodate large insert protein
Simultaneously express multiple genes
Safer to use than mammalian virus
…
Mammalian Expression System
Plasmid Transfection
Viral Vector Infection
Mammalian Expression System
Transient Expression System:Short Protein Expression Duration Simple Short Time
Stable Cell Line Generation:Time-consuming and laboriousProtein Expression Last a Long Time
Mammalian Expression System
Advantages:
Initiation Signals
Protein Processing
Protein Secretion
Protein Glycosylation
Intact Macromolecules
Native Structure
…
Disadvantages:
Complicated
High Requirement
Low Yield
Sometimes Viral Infections
…
Bacterial Expression
SystemYeast Expression System
Insect Expression System
Mammalian Expression System
Speed ★★★★ ★★★ ★★ ★
Yield ★★★ ★★★★ ★★ ★
PTM(relative to
human)★ ★★ ★★★ ★★★★
Cost ★★★★ ★★★ ★★ ★
Application
Prokaryotic protein, simple
eukaryotic protein
Intracellular/Secreted Protein, Disulfide-bonded
protein, Glycosylated protein
Membrane protein, Large-size protein,
Viral vaccines, signaling proteins, cytokines, kinases
Complex eukaryotic protein, Protein need
accurate PTM
Simply Compare among Expression Systems
Contact Information:
Phone: 1-631-559-92691-631-448-7888
Fax: 1-631-938-8127 Address: 45-1 Ramsey Road, Shirley,
NY 11967, USA Email: [email protected]
Thanks!Any questions?You can find me at [email protected]