Title: Twostage amplification of induction:
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2Inducible expression in E. coli T7 system
Two-stage amplification of induction Many T7 Pol
molecules from one T7 pol gene Many YFG molecules
for every T7pol molecule Does not need to compete
with E. coli RNA pol all the T7 pol available
for transgene transcription.
plasmid
T7 prom.
X
T7 RNA polymerase
chromosome
IPTG
T7 RNA polymerase
Lac promoter
IPTG
X
Constitutive synthesis
chromosome
T7 RNA polymerase
Lac promoter
Lac I (repressor)
3IPTG -------gt T7 RNA pol ---------gt His6- cDNA
4OFF
IPTG
ON
5His tag allows easy purification of the cloned
gene product
(Nitriloacetic acid chelate, NTA)
(competes with his)
Other cell proteins
6Assorted protein tags used for purification
harmful
(from Protein A)
(binds biotin)
(biotinylated)
(poly-glucose)
(tripeptide)
Protein A is a bacterial protein that binds
tightly to IgG Streptavidin binds biotin
strongly (Kd10-15). MBP maltose binding
protein GST glutathione-S-transferase
7cDNA
VECTOR
Enterokinase Asp Asp Asp Asp Lys
8Half-genes
Linearized viral genome
Gene of interest
Strong polyhedron promoter
(Fall army worm)
Baculovirus as a vector
in insect cells
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12Tn7 transposase
Transposition/recombination between Tn7
sites disrupts lacZ gene
13Production of recombinant baculovirus
14Yeast Expression Vector
2mm-seq yeast ori oriE bacterial ori Ampr
bacterial selection LEU2 Leu biosynthesis
yeast selection
2mm plasmid
Gene ofinterest(GOI)
Complementation of an auxotrophy can be used
instead of drug-resistance
Saccharomyces cerevisiae(bakers yeast)
15Yeast -Genomic integration
HIS4
t
p
YFG
FunctionalHIS4 gene
DefectiveHIS4 gene
16Double recombination Yeast (integration in Pichia
pastoris)
P. pastoris-tight control-methanol induced
(AOX1)-large scale production (gram
quantities)
Alcohol oxidase gene
17Mammalian cell expression Popular hosts cell
lines HeLa (human cevial cancer) HEK293 (human
embryonic kidney Vero (monkey) Cos
(monkey) Mouse 3T3 CHO (Chinese hamster ovary)
BHK (baby hamster kidney)
polyA site
intron
cDNA gene
Mam. prom.
3UTR
5UTR
18Expression in mammalian cells HEK293, HeLa, CHO,
cos, Popular mammalian cell promoters
- SV40 LargeT Ag (Simian Virus 40)
- RSV LTR (Rous sarcoma virus)
- MMTV (steroid inducible) (Mouse mammary tumor
virus) - HSV TK (low expression) (Herpes simplex virus)
- Metallothionein (metal inducible, Cd)
- CMV early (Cytomegalovirus)
- Inducible / repressible tet, ecdysone,
glucocorticoid (tet tetracycline)
19In vitro translation following in vitro
transcription
Add energy source, tRNAs, amino acids (label),
RNA (remove endogenous mRNA)
VECTOR
cDNA
T7 RNA pol Binding site
ACCATGG..
ACCATGG..
20Biochemistry of purified protein in vitro
Binding between (almost) purified
proteins- His6- X HA- Y Bind to Nickel
ion column, elute, Western with HA Ab His6-
X 35S- Y (made in vitro) Bind Ni column,
elute, gel autorad.
BIAcore (or several other measurements)
In cells- co-IP from extracts, FRET, 2-hybrid
21SURFACE PLASMON RESONANCE
1- Glass 2- Gold 3- Polymer Circle- Protein Y-
Binding protein
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23Flow cell carrying ligands or wash
24Co-immunoprecipitation Use protein A immobilized
on beads (e.g., agarose beads) Protein A from
Staphylococcus aureus binds tightly to
Immunoglobulin G (IgG) from many species.
anti-X IgG
Cell extract
Protein A
Wash by centrifugation Elute with SDS Detect X, Y
by Western blotting
25FRET
http//mekentosj.com/science/fret/fret.html
26FRET Fluorescence resonance energy transfer
(Excite with this)
Emission at 530 nm
(Direct, no photons)
(measure this)
Excitation At 530 nm
The closer the fluorophores are to each other,
the greater the FRET Distances up to 100 A can
be detected Changes down to 2 A can be
measured Intramolecular distances and their
changes can be measured with FRET Usually
measured in a fluorometer FRET can be seen in a
fluorescent microscope
Emission at 570 nm
YFP Yellow fluorescent protein CFP Cyan
fluorescent protein
27FRET example The role of particular proteins in
regulated actin remodellingat the edge of a cell
membrane
Blue light in, Green out Green light in,
Red out Blue light in, red out FRET
Diaphanous-related formins (Drfs) act as Rho
small GTPase effectors during factor-induced
cytoskeletal remodelling.
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29In vitro Site-specific mutagenesis
30Dut deoxyuracil phophotransferase. Dut- cells
accumulate dUTP--gt DNA Ung uracil
N-glycosylase. Ung- mutants do
not excise uracil from DNA Growth of plasmid
template plasmid on dut- ung- E. coli double
mutant cells gives U in DNA Synthesize mutant
DNA using Transfect heteroduplex into dutung
E. coli. U gets removed from WT template strand,
leaving apyrimidinic sites, these cannot
replicate. Mutant synthetic strand survives
normally.
31PCR-ligation-PCR mutagenesis
Site to mutate
Note you can easily create an internal deletion
this way.
Ali SA, Steinkasserer A., Biotechniques. 1995
May18(5)746-50.
32RS1
RS2
Mutagenesis by overlap extension PCR
PCR ? fragment ? subsequent cloning in a plasmid
RS1
RS2
Cut with RE 1 and 2
Ligate into similarly cut vector
33Quik-change
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