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. Lac I (repressor) Constitutive synthesis T7 RNA polymerase Lac promoter X +IPTG T7 RNA polymerase Lac promoter IPTG X T7 prom. plasmid 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 IgG Streptavidin binds biotin strongly (Kd~ ). MBP = maltose binding protein GST = glutathione-S-transferase Assorted protein tags used for purification (binds biotin) (biotinylated) (from Protein A) harmful (poly-glucose) (tripeptide)

cDNAVECTOR EnterokinaseAsp Asp Asp Asp Lys ……

Baculovirus as a vector in insect cells Half-genes (Fall army worm) Strong polyhedron promoter Gene of interest Linearized viral genome

Tn7 transposase Transposition/recombination between Tn7 sites disrupts lacZ gene

Production of recombinant baculovirus

Yeast Expression Vector 2  m plasmid 2  m-seq = yeast ori ori E = bacterial ori Amp r = bacterial selection LEU2 = Leu biosynthesis yeast selection Saccharomyces cerevisiae (baker’s yeast) ori E Gene of interest (GOI) LEU2 Amp r GAPDterm GAPDprom Complementation of an auxotrophy can be used instead of drug-resistance

Genomic DNA HIS4 - Yeast -Genomic integration HIS4 YFG pt Vector DNA Functional HIS4 gene Defective HIS4 gene YFG t p Genomic DNA

Double recombination Yeast (integration in Pichia pastoris) AOX1 gene (<= 30% of protein) Genomic DNA AOX1p GOI AOX1tHIS4 3’AOX1 Genomic DNA HIS4 GOI 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 kidney Vero (monkey) Cos (monkey) Mouse 3T3 CHO (Chinese hamster ovary) BHK (baby hamster kidney) cDNA gene Mam. 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 Lysate E.coli lysate Add energy source, tRNAs, amino acids (label), RNA (remove endogenous mRNA) cDNA T7 RNA pol Binding site 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}

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. 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 centrifugation Elute with SDS Detect X, Y by Western blotting Cell extract

FRET

FRET: Fluorescence resonance energy transfer YFP = Yellow fluorescent protein CFP = Cyan fluorescent protein Emission at ~530 nm Excitation At 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 remodelling at the edge of a cell membrane Blue light in, Green out Green light in, Red out Blue light in, red out = FRET

28 Reporter Binds DNA Bait- test protein Activates Transcription locally Candidate Binding protein Yeast Two Hybrid Assay

29

30 Single bait vectorLibrary of cDNAs in target vector Already in yeast with Reporter genes Transform into yeast

31

32 lacZ for color HIS3 for growth