two-stage amplification of induction: many t7 pol molecules from one t7 pol gene many yfg molecules...
TRANSCRIPT
Two-stage amplification of induction:Many T7 Pol molecules from one T7 pol geneMany YFG molecules for every T7pol moleculeDoes not need to compete with E. coli RNA pol; all the T7 pol available for transgene transcription.
Lac I (repressor)
Constitutive synthesis
T7 RNA polymerase
Lac promoter
X
+IPTG
T7 RNA polymerase
Lac promoter
IPTG
XT7 prom.
plasmid
chromosome
chromosome
Inducible expression in E. coli: T7 system
T7 RNA polymerase
IPTG -------> T7 RNA pol ---------> His6- cDNA
OFF
ON
+ IPTG
(competes with his)
His tag allows easy purification of the cloned gene product
(Nitriloacetic acid chelate, NTA)
Other cell proteins
Protein A is a bacterial protein that binds tightly to IgGStreptavidin binds biotin strongly (Kd~10-15). MBP = maltose binding proteinGST = glutathione-S-transferase
Assorted protein tags used for purification
(binds biotin)
(biotinylated)
(from Protein A) harmful
(poly-glucose)
(tripeptide)
cDNAVECTOR
Enterokinase Asp Asp Asp Asp Lys ……
Baculovirus as a vectorin insect cells
Half-genes
(Fall army worm)
Strong polyhedron promoter
Gene of i
ntere
st
Linearizedviral genome
Tn7 transposase
Transposition/recombination between Tn7 sitesdisrupts lacZ gene
Production of recombinant baculovirus
Yeast Expression Vector
2m plasmid
2m-seq = yeast ori
oriE = bacterial ori
Ampr = bacterial selection
LEU2 = Leu biosynthesis yeast selection
Saccharomyces cerevisiae(baker’s yeast)
oriE
Gene ofinterest(GOI)
LEU2
Ampr
GAPDterm
GAPDprom
Complementation of an auxotrophy can be used instead of drug-resistance
Genomic DNA
HIS4-
Yeast -Genomic integration
HIS4
YFG
pt Vector DNA
FunctionalHIS4 gene
DefectiveHIS4 gene
YFG
tp
Genomic DNA
Double recombination Yeast (integration in Pichia pastoris)
AOX1 gene (<= 30% of protein)
Genomic DNA
AOX1p
GOI
AOX1t HIS4 3’AOX1
Genomic DNA
HIS4
GO
I
AOX1p
AOX1t
3’AOX1
Vector DNA
P. pastoris-tight control-methanol induced (AOX1)-large scale production (gram quantities)
Alcohol oxidase gene
Mammalian cell expression
Popular hosts cell lines:
HeLa (human cevial cancer)HEK293 (human embryonic kidneyVero (monkey) Cos (monkey)Mouse 3T3CHO (Chinese hamster ovary) BHK (baby hamster kidney)
cDNA geneMam. prom.
polyA site
intron
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 LysateE.coli lysate
Add energy source, tRNAs, amino acids (label), RNA (remove endogenous mRNA)
cDNA
T7 RNA polBinding site
ACCATGG…..
ACCATGG…..
VECTOR
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}
SURFACEPLASMONRESONANCE
1- Glass2- Gold3- PolymerCircle- ProteinY- 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.
A
A
A
A
A
A
X
Y
A
Y
X
C
B
X
Y
+
A
Y
X
C
B
+ anti-X IgG
A
A
A
A
A
A
X
Y
+
A
Y
X C
B
+ Protein A
A
A
A
A
A
A
Y
X
X
Y
Wash by centrifugationElute with SDSDetect X, Y by Western blotting
Cell extract
FRET
http://mekentosj.com/science/fret/fret.html
FRET: Fluorescence resonance energy transfer
YFP = Yellow fluorescent proteinCFP = Cyan fluorescent protein
Emission at ~530 nm
ExcitationAt 530 nm
Emission at ~570 nm
(Direct, no photons)
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
(measure this)
(Excite with this)
Diaphanous-related formins (Drfs) act as Rho small GTPase effectors during factor-induced cytoskeletal remodelling.
FRET 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
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Reporter
BindsDNA
Bait- test protein
ActivatesTranscriptionlocally
CandidateBindingprotein
Yeast Two Hybrid Assay
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Single bait vector Library of cDNAs in target vectorAlready in yeast with
Reporter genes Transform into yeast
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lacZ for colorHIS3 for growth