Inducible expression in E coli T 7 system
Inducible expression in E. coli: T 7 system plasmid Two-stage amplification of induction: Many T 7 Pol molecules from one T 7 pol gene Many YFG molecules for every T 7 pol molecule Does not need to compete with E. coli RNA pol; all the T 7 pol available for transgene transcription. T 7 prom. X T 7 RNA polymerase IPT G Lac promoter T 7 RNA polymerase chromosome +IPTG Constitutive synthesis Lac I (repressor) X Lac promoter T 7 RNA polymerase chromosome
IPTG -------> T 7 RNA pol -----> His 6 - c. DNA
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 Ig. G Streptavidin binds biotin strongly (Kd~10 -15). MBP = maltose binding protein GST = glutathione-S-transferase
VECTOR Enterokinase c. DNA Asp Asp Lys ……
Half-genes Linearized viral genome ne e G e nt i of st e r Strong polyhedron promoter (Fall army worm) Baculovirus as a vector in insect cells
Tn 7 transposase Transposition/recombination between Tn 7 sites disrupts lac. Z gene
Production of recombinant baculovirus
Yeast Expression Vector 2 mm-seq = yeast ori 2 mm plasmid ori. E = bacterial ori Ampr = bacterial selection GAPDterm LEU 2 = Leu biosynthesis yeast selection Complementation of an auxotrophy can be used instead of drug-resistance Gene of interest (GOI) LEU 2 Saccharomyces cerevisiae (baker’s yeast) Ampr GAPDprom ori. E
Yeast -Genomic integration t Vector DNA p YFG HIS 4 Genomic DNA HIS 4 t p YFG Functional HIS 4 gene Defective HIS 4 gene
Double recombination Yeast (integration in Pichia pastoris) P. pastoris -tight control -methanol induced (AOX 1) -large scale production AOX 1 t (gram quantities) HIS 4 Vector DNA GOI AOX 1 p 3’AOX 1 Genomic DNA Alcohol oxidase gene AOX 1 gene (<= 30% of protein) Genomic DNA GOI AOX 1 p AOX 1 t HIS 4 3’AOX 1
Mammalian cell expression Popular hosts cell lines: He. La (human cevial cancer) HEK 293 (human embryonic kidney Vero (monkey) Cos (monkey) Mouse 3 T 3 CHO (Chinese hamster ovary) BHK (baby hamster kidney) poly. A site Mam. prom. intron 5’UTR c. DNA gene 3’UTR
Expression in mammalian cells: HEK 293, He. La, CHO, cos, Popular mammalian cell promoters • • SV 40 Large. T 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, t. RNAs, amino acids (label), RNA (remove endogenous m. RNA) VECTOR c. DNA T 7 RNA pol Binding site ACCATGG…. .
Biochemistry of purified protein in vitro Binding between (almost) purified proteins: His 6 - X + HA- Y ; Bind to Nickel ion column, elute, Western with HA Ab His 6 - X + 35 S- 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 (Ig. G) from many species. B Y X Y Y X + C A X B nti -X C X Y Cell extract +a Ig. G A A Y A A A X A A A A X Y + Protein A Y X A A Wash by centrifugation A Elute with SDS Detect X, Y by Western blotting A A B + C X Y A
FRET http: //mekentosj. com/science/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 c. DNAs in target vector Transform into yeast 30
31
lac. Z for color HIS 3 for growth 32
- Slides: 32