Two-stage amplification of induction: Many T7 Pol molecules from one T7 pol gene Many YFG molecules...

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

29

30

Single bait vector Library of cDNAs in target vectorAlready in yeast with

Reporter genes Transform into yeast

31

32

lacZ for colorHIS3 for growth