Download Topic 6

Inducible expression in E. coli: T7 system
plasmid
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.
T7 prom.
X
T7 RNA
polymerase
chromosome
Lac promoter
T7 RNA
polymerase
+IPTG
Constitutive synthesis
chromosome
Lac I (repressor)
Lac promoter T7 RNA
polymerase
IPTG -------> T7 RNA pol ---------> His6- cDNA
OFF
+ IPTG
ON
His tag allows easy purification of the cloned gene product
(Nitriloacetic acid
chelate, NTA)
(competes with his)
Other cell proteins
Assorted 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 (Kd~10-15).
MBP = maltose binding protein
GST = glutathione-S-transferase
VECTOR
Enterokinase
cDNA
Asp Asp Asp Asp Lys ……
Half-genes
Linearized
viral genome
Strong polyhedron
promoter
(Fall army worm)
Baculovirus as a vector
in insect cells
Tn7 transposase
Transposition/recombination between Tn7 sites
disrupts lacZ gene
Production of recombinant baculovirus
Yeast Expression Vector
2mm-seq = yeast ori
oriE = bacterial ori
2mm plasmid
Ampr = bacterial selection
GAPDterm
LEU2 = Leu biosynthesis
yeast selection
Complementation of
an auxotrophy can
be used instead of
drug-resistance
Gene of
interest
(GOI)
LEU2
Saccharomyces cerevisiae
(baker’s yeast)
Ampr
GAPDprom
oriE
Yeast -Genomic integration
t
p
Vector
DNA
YFG
HIS4
Genomic
DNA
Genomic
DNA
HIS4-
t
p
YFG
Functional
HIS4 gene
Defective
HIS4 gene
Double recombination Yeast (integration in Pichia pastoris)
P. pastoris
-tight control
-methanol induced (AOX1)
-large scale production
AOX1t
(gram quantities)
HIS4
Vector
DNA
GOI
3’AOX1
AOX1p
Genomic
DNA
Alcohol oxidase gene
AOX1 gene (<= 30% of protein)
Genomic
DNA
GOI
AOX1p
AOX1t
HIS4
3’AOX1
Mammalian 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
Mam. prom.
intron
cDNA gene
5’UTR
3’UTR
Expression 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)
In vitro translation following in vitro transcription
Rabbit Reticulocyte lysate
Wheat Germ Lysate
E.coli lysate
Add energy source, tRNAs, amino acids (label),
RNA (remove endogenous mRNA)
VECTOR
cDNA
T7 RNA pol
Binding site
ACCATGG…..
ACCATGG…..
Biochemistry 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}
SURFACE
PLASMON
RESONANCE
1- Glass
2- Gold
3- Polymer
Circle- Protein
Y- Binding protein
glass plate
Flow cell carrying ligands or wash
Co-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.
Y
B
X
X
Y
Y
B
+
C
A
C
X
X
Y
Cell extract
A
A
Y
A
A
A
X
X
Y
Y
X
+
A
A
A
A
A
A
A
A
A
B
+ Protein A
A
C
A
A
Wash by centrifugation
A
Elute with SDS
Detect X, Y by Western blotting
A
X
Y
A
FRET
http://mekentosj.com/science/fret/fret.html
FRET: Fluorescence resonance energy transfer
(Excite with this)
Emission at ~530 nm
(Direct,
no photons)
Excitation
At 530 nm
(measure this)
The closer the fluorophores are to
each other, the greater the FRET
Distances up to 100 A can be detected
Emission at ~570 nm
Changes down to 2 A can be measured
Intramolecular distances and their
changes can be measured with FRET
Usually measured in a fluorometer
YFP = Yellow fluorescent protein
CFP = Cyan fluorescent protein
FRET can be seen in a fluorescent
microscope
FRET example: The role of particular proteins in regulated actin remodelling
at 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.
Yeast Two Hybrid Assay
Binds
DNA
Baittest protein
Candidate
Binding
protein
Activates
Transcription
locally
Reporter
28
29
Single bait vector
Already in yeast with
Reporter genes
Library of cDNAs
in target vector
Transform into yeast
30
31
lacZ for color
HIS3 for growth
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