1. Cloning Club: Multiple proteins from one plasmid
Merrifield 163, first sythetic protein 1969
Bacterial expression construct

2. Cloning Club: Multiple proteins from one plasmid
You can clone an E. coli expression vector like this
(multicistronic expression):
Merrifield 163, first sythetic protein 1969
But why can’t you clone a mammalian vector like this?

3. The lac operon (3 genes under one promoter)

4. Initiation of translation
RBS
(= Shine-Dalgarno sequence)
5-9 nt
ATG: 80%
CTG: 15%
TTG&GTG: 5%
E. coli
UAAGGAGG
Mammalian cells
5’-cap
Kozak sequence
The 5’-cap is (mostly)
needed for the
initiation of scanning
Cap-independent
Translation → IRES

5. Cloning Club: Multiple proteins from one plasmid
GOI 1
GOI 2
Promoter 1
Promoter 2
GOI 1
GOI 2
Promoter
IRES
internal ribosome entry site (IRES)
GOI 1
GOI 2
Promoter
CTG
ATG
non-AUG initiation
GOI 2
Promoter
GOI 1
G↔A
2A element
Merrifield 163, first sythetic protein 1969
GOI 2
Promoter
intron
intron
Prom.
GOI 1
intron
intron
intronic expression
GOI 2
Promoter
GOI 1
modified intein
intron
„protein intron“
Ratio of expression between GOI1 and GOI2 can be regulated: IRES < cap-dependent translation, CTG << ATG

6. Baculovirus System More than one Gene Of Interest (GOI)
2 GOIs: pFastBac Dual (LifeTechnologies)
Many GOIs: MultiBac (Geneva Biotech)

7. Baculovirus System The same plasmid for bacterial,
baculoviral and mammalian expression
pTriEx (Novagen→Merck/Millipore)
pQE-TriSystem (Qiagen)

8. Next meeting Topic: Gateway and Golden Gate Cloning
Again in room KOK7 (7th floor)