Download Expression system

Prokaryotic Expression Systems
• E. coli, Bacillus subtilis, Staphylococcus carnosus,
Streptomyces lividans
• Prokaryotic promoter—ribosome binding site—MCS—
transcription termination site
• Prokaryotic selectable marker
Fig.1
Expression of cloned genes
in bacteria Expression
vectors contain promoter
sequences (pro) that direct
transcription of inserted DNA
in bacteria and sequences
required for binding of mRNA
to bacterial ribosomes (ShineDelgarno [SD] sequences). A
eukaryotic cDNA inserted
adjacent to these sequences
can be efficiently expressed in
E. coli, resulting in production
of eukaryotic proteins in
transformed bacteria.
Necessary Features of expression vector:
•Origin of replication
•Drug resistance marker
•A promoter
•Transcription terminator
•Restriction sites Expression
Vectors with or without specific tags……
• Vectors for non-fusion proteins pKK223-3 pSVK 3 PSVL
SV40
• Vectors for fusion proteins pGEX pQE pET pEZZ 18
Promoters of Importance in Biotech
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lac
trp
tac (or trc)
phage l pL
phage T7 gene 10
Fusion proteins
• Used to avoid problems with digestion of foreign
proteins by proteases
• Used to aid purification of foreign proteins, often by
affinity chromatography
• Often a rare protease cut site is added to the fusion
partner
• Eg., For small peptides poly-arginine,Histidine tail
• Large peptides Maltose binding protein( MBP) Chitin
binding domain Cellulose binding domain Glutathione
S- transferase( GST)
Features of Host --prokaryotic systems ex: E coli
Advantages
Disadvantages
•Many references and much
experience available e.g.
E. coli.
•Wide choice of cloning
vectors.
•Gene expression easily
controlled.
•Easy to grow with high
yields.
•Product can be designed
for secretion into the
growth media.
• Low expression
• No post-translational
modification
• Degradation by bacterial
proteases
• . •Biological activity and
immunogenicity may
differ from natural protein.
•High endotoxin content in
gram negative.
Eukaryotic Expression Systems
• Yeast, Aspergillus niger, Baculovirus-Insect Cells,
Mammalian Cells (e.g. Chinese Hamster Ovary cells)
• Eukaryotic promoter-MCS-transcription termination site
• Eukaryotic selectable marker
Fig. 2
Features of eukaryotic systemsyotic : Yeast
Advantages
• Unlike prokaryotic expression systems, allow for
posttranslational modification (PTM) of proteins (e.g.,
glycosylation, phosphorylation, hydroxylation, disulfide
bond formation, processing of propeptides)
•Lacks detectable endotoxins.
•Fermentation relatively inexpensive.
Disadvantage
• Expression less easily controlled.
• Glycosylation not identical to mammalian systems.