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• synthesis and purification of proteins
• Proteins produced in two types:
– Native proteins
– Fusion proteins
• We need – Appropriate promoter
– Appropriate host• Prokaryotic host
– E.Coli
– B. Subtilis
• Eukaryotic– Yeast
– Fungi
– Insect
– Mamalian cell line
– Transgenic animal
Cloning and Expression OptimizationDominic Esposito, Group Leader
Cloning•PCR Gateway Sequencing•Restriction enzymes and ligase•Multiple expression vectors (>50)
Expression optimization (E. coli)•Strains•Temperature•Fusion tags•Induction time, level
Microbial FermentationDavid Miller, Group Leader
•E. coli, yeast•Shake flasks to 60 liters•Batch and fed batch•Cell lysis and processing (membranes)•Isotopic labeling
Microbial Fermentation:• Computer controlled 80 liter fermenter, workingvolume 20 – 60 liters
• 4 x 20 liter fermenters, computer controlled, 5 –15 liter working volumes
Yeast systems
• Saccharomyces cerevisiae
• Schizosaccharomyces pombe
• Pichia pastoris
• Hansela polymorpha
• Kluyveromyces lactis
• Yarrowia lipolytica
S. Serviciae or
GRAS (Generally Regarded As
Safe)• Intracellular expression
– higher protein yields
– But• more difficult extraction
• and purification.
• co- and post-translational processing of proteins at N- and C-termini.
• proteolytic degradation
• addition of tags might result in aggregation and insolubility
• Secretion
– N-terminal signal sequences for co-translational translocation
– Then removed by a signal peptidase
– Examples of popular signal sequences• Pho5, Suc2 and the a -factor.
• Vectors based on a small multi-copy plasmid
(called the 2μ plasmid)• YEps (for yeast episomal plasmids)
• Use auxotrophic host
Disadvantage of yeasts
• Hyperglycosylation which leads to • Reduce activity
• Immunogenicity
– To solve this problem
• Glycosylation mutants
• Remove of glycosylation sites
• Using of some other type of yeast such as P. pastoris
P.pastoris
• highly developed fermentation technology
• can be grown to higher cell densities in culture and in fermentors
• it can secrete proteins to much higher levels than S. cerevisiae
• Yields of heterologous proteins of around 12g/L in P.pastoris
• Use methanol as carbon source
• alcohol oxidase promoter (AOX1) for heterologous protein expression by adding of methanol to medium
• This promoter strongly repressed in absence of methanol
• Integrative and autonomously replicating (PARS1 and PARS2) vectors available
• Protein can produce by intracellular expression or secretion
• A better secretor than S.c
• Glycoproteins are often less mannosylated than those, of Saccharomyces
• About 35% of N-linked oligosaccharides have less than 14 mannose units
• Pichia's secretion signal from its own acid phosphatase
• Proteolysis in Pichia– Can be minimised by buffering the pH of the medium
Baculovirus system
• Uses insect cells from Spodopterafrugiperda
• infected with baculoviruses Autographa
californica
• Use polyhedrin promoter to expression
Baculovirus Vector
• Vector has sequences for expression using po
• lyhedron gene expression system
• Sequences also present for integration into baculovirus
(AcMNPV) genome via recombination
• Prokaryotic sequences not shown
E. coli Baculovirus Shuttle
Vector - Bacmids
• Shuttle vectors allow ease of transfer between systems
• Genetic manipulations in one system, expression in another
Bacmid Construction (2)
• attR and attL are lambda sequences to give high
efficiency and specific transposition
Modifying the Insect Cell Host
• Some enzymes simply not present
• Genetic engineering of the host for proper
expression
• Add missing glycosylation enzymes
• Add proteolytic processing enzymes
AdvantagesThe polyhedrin gene is not required for the continuous production of infectious virus in
insect cell culture. Its sequence is replaced with that of the heterologous gene.
The polyhedrin gene promoter is very strong. This determines a very high level of
production of recombinant protein.
Very late expression allows for the production of very toxic proteins.
This system is capable of post-translational modifications.
Disadvantages
•Expensive.
•Glycosylation in insect cells is different (insect cells unable to produce complex N-linked
side chains with penultimate galactose and terminal sialic acid) from that in vertebrate
cells, therefore, a problem for therapeutic proteins.
•A large fraction of the RP can be poorly processed and accumulates as aggregates.
•Discontinuous expression: baculovirus infection of insect cells kills the host and hence
the need to reinfect fresh cultures for each round of protein synthesis.
•Inefficient for production on a commercial scale.
Mammalian Systems
• Sometimes insect cells simply don’t carry
out proper/necessary glycosylations
• Other processing may also not occur
• Mammalian cell systems are more
expensive by may be required for active
product
Mammalian Expression Vector
• “I” is an intron that enhances expression
• Other signals similar to insect and prokaryotic
vectors
Translation Control Elements
• K - Kozak Sequence (equiv. To rbs)
• S - for secretion signal peptide
• T – tag peptide for purification
• P – proteolytic cleavage sequence
• SC – stop codon for translation
• 3’UTR – proper sequences for efficient translation and
mRNA stability (e.g. polyadenylation sequence)
Bicistronic Expression Vector
IRES from mammalian virus
•Gives more uniform level of expression of two genes
Selectable Markers for Mammalian
Systems
• Most commonly used to select for
transformed cells (killing nonresistant ones)
• Can be used for increasing expression of
heterologous proteins
Use of Selectable Markers for
Increasing Heterologous Protein
Production in Mammalian Systems
• Methotrexate (MTX ) inhibits dihydrofolate
reductase (DHFR)
• DHFR- host cell with DHFR gene on cloning
vector (i.e. linked to target gene)
• Gradually increase MTX concentration in culture
• Gene copy number of DHFR and linked target
gene increase to compensate for inhibition of
DHFR (more protein that is less active gives cell
enough metabolic through put to survive)