Presentation on theme: "11 1 Yeast Expression Vector (example) 2μ = 2 micron plasmid 2 mu seq features: yeast ori ori E = bacterial ori Amp r = bacterial selection LEU2, e.g."— Presentation transcript:
11 1 Yeast Expression Vector (example) 2μ = 2 micron plasmid 2 mu seq features: yeast ori ori E = bacterial ori Amp r = bacterial selection LEU2, e.g. = Leu biosynthesis for yeast selection Saccharomyces cerevisiae (baker’s yeast) ori E Your favorite gene (Yfg) LEU2 Amp r GAPD term’n GAPD prom Complementation of an auxotrophy can be used instead of drug-resistance Auxotrophy = state of a mutant in a biosynthetic pathway resulting in a requirement for a nutrient GAPD = the enzyme glyceraldehyde-3 phosphate dehydrogenase For growth in E. coli Nov. 8, :00 AM
22 Genomic DNA HIS4 mutation - Yeast - genomic integration via homologous recombination HIS4 gfY pt Vector DNA Functional HIS4 gene Defective HIS4 gene Yfg t p Genomic DNA
33 Double recombination Yeast (integration in Pichia pastoris) AOX1 gene ( ~ 30% of total protein) Genomic DNA AOX1p Yfg AOX1tHIS4 3’AOX1 Genomic DNA HIS4 Yfg AOX1p AOX1t 3’AOX1 Vector DNA P. pastoris -tight control -methanol induced (AOX1) -large scale production (gram quantities) Alcohol oxidase gene
4 Primary cells cultured with a limited lifetime. E.g., MEF = mouse embryonic fibroblasts, HDF = Human diploid fibroblasts Mammalian cell lines (lines implies immortal) Primary culture: human cells < 50 generations (doublings). Then senescence. Low frequency of survivors, increased by mutagens (carcinogens) Mouse cells earlier senescence, higher frequency of survivors Human cells + 3 exogenous genes tumor cells (ras, SV40 T, telomerase) (Hahn et al., Creation of human tumour cells with defined genetic elements. Nature :464-8)Nature. Cell lines are typically aneuploid (abnormal number of and rearranged chromosomes). Often sub-tetraploid in number (human diploid chroosome number = 46, HeLa cells ~69, or 82 etc. variable).
5 Expression in mammalian cells Lab examples of immortal cell lines: HEK293 Human embyonic kidney (high transfection efficiency) HeLa Human cervical carcinoma (historical, low RNase) CHO Chinese hamster ovary (hardy, diploid DNA content, mutants) CosMonkey cells with SV40 replication proteins (-> high transgene copies) 3T3Mouse or human exhibiting ~regulated (normal-like) growth + various others, many differentiated to different degrees, e.g.: BHKBaby hamster kidney HepG2Human hepatoma GH3Rat pituitary cells PC12Mouse neuronal-like tumor cells MCF7Human breast cancer HT1080 Human fibroblastic cells with near diploid karyotype IPSinduced pluripotent stem cells and: Common in industry for production: NS1mAbsMouse plasma cell tumor cells Vero vaccines African greem monkey cells CHOmAbs, other therapeutic proteinsChinese hamster ovary cells PER6mAbs, other therapeutic proteinsHuman retinal cells
6 Mammalian cell expression Generalized gene structure for mammalian expression: cDNA gene Mam.prom. polyA site intron 5’UTR 3’UTR Intron is optional but a good idea
8 Engineered regulated expression: Tetracycline-reponsive promoters Tet-OFF (add tet shut off) tTA cDNA tTA = tet activator fusion protein: tetR = tet repressor (original role) tetR domain VP16 transcription activation domain No tet. Binds tet operator (multiple copies) (if tet not also bound) tetR domain Tetracycline (tet), or, better, doxicyclin (dox) active not active CMV prom. polyA site tTA gene must be in cell (permanent transfection, integrated): Tet-OFF (Bujold et al.) Allosteric change in conformation VP16 transcription activation domain
9 MIN. CMV prom. your favorite gene polyA site Mutliple tet operator elements MIN. CMV prom. your favorite gene polyA site tetR domain VP16 tc’n act’n domain not active little transcripton (2%?, bkgd) Doxicyclin present: MIN. CMV prom. your favorite gene polyA site active Plenty of transcripton No doxicyclin: tetR domain VP16 tc’n act’n domain RNA po l Tet-OFF, cont.
10 Tetracycline-reponsive promoters Tet-ON (add tet turn on gene tTA cDNA tetR domain VP16 tc’n act’n domain tetR domain VP16 tc’n act’n domain Tetracycline (tet), or, better, doxicyclin (dox) active not active Full CMV prom. polyA site Different fusion protein: Does NOT bind tet operator (if tet not bound) Tet-ON Must be in cell (permanent transfection, integrated): commercially available (293, CHO) or do-it-yourself
11 MIN. CMV prom. your favorite gene polyA site Mutliple tet operator elements MIN. CMV prom. your favorite gene polyA site active Doxicyclin absent: MIN. CMV prom. your favorite gene polyA site active Plenty of transcripton (> 50X) Add dox: tetR domain VP16 tc’n act’n domain RNA pol II Tet-ON tetR domain VP16 tc’n act’n domain not active little transcription (bkgd.) doxicyclin
12 Biotechnology methods to study transcriptional regulation in cells Mainly, use of reporter proteins whose cDNA sequence is linked to the promoter. First, a synopsis of promoter structure:
13 General model for transcriptional regulation in higher eukaryotes TF… transcription factor TBP: TATA binding protein TAF: TBP associated protein BRE: TFIIB response element INR: transcription initiator element DPE: downstream promoter element The transcription complex either recruits RNA Pol II or activates a bound RNA Pol II Core transcriptional elements For review see Smale and Katonga, Ann. Rev. Biochem. 72: (2003) GGGCGCC; CCACGCC TATA(AT)AA(GA) YYAN(TA)YY Y = C or T (pyrimidine) (AG)G(AT)(CT)(GAC)
14 Many transcriptional enhancer elements often lie upstream of promoters, allowing for many combinations of TF binding
15 Put a DNA regulatory region upstream of a reporter gene to analyze its elements PCR Space for res. enz. to bind Reporter gene Transfect
16 Popular reporters to study promoter/enhancers Beta-galactosidase (β-gal) – detection by several different assays Chloramphenicol acetyl transferase (CAT) – detection, sensitive radioactive assay Luciferase (firefly, Renilla [jellyfish]) – detection, easy dual, sensitive luminescent assay Green fluorescent protein (GFP, BFP, YFP)) – cytological, visible in living cells, fusion proteins, FACS Neomycin phosphotransferase (neo)–selectable drug resistance (G418 R ) (similarly: resistance to hygromycin, puromycin, histidinol, zeocin) Dihydrofolate reductase (DHFR) – selectable in dhfr- cells, amplifiable, fusion proteins work Suicide selection: Herpes simplex virus thymidine kinase (HSVTK) FACS = fluorescence-activated cell sorter
17 diacetylated monoacetylated Testing for a cell-specific promoter: chloramphenicol acetyl transferase (CAT) reporter assay (www.biochem.arizona.edu/classes/bioc471/pages/Lecture15/Lecture15.html) Thin layer chromatography (TLC) CAT cDNA is from a prokaryotic source. CAT is not found in mammalian cells. Therefore low backgrounds A B 14 C-chloramphenicol unacetylated Positive control Negative control
18 Reporter enzyme substrates for different purposes ONPG (ortho-nitrophenyl-beta-galactoside) – spectrophotometric measurement (420 nm – blue color – simplest) X-gal (5-Bromo-4-chloro-3-indolyl-ß-D-galactoside) – blue precipitate - for cytology or colony detection Umbelliferyl–galactoside (-> umbelliferone, fluorescent, reading in a fluorimeter allows more sensitive quantification than spectrophotometry) Galacton-STAR or some such (-> chemiluminescent product = emission of light, so lower background than fluorescence) Lactose (glucose-beta-galactose disaccharide) – allows growth if hydrolyzed; growth phenotype. For microbial cells usually. Substrates for beta-galactosidase, for example:
19 Mapping transcriptional elements upstream of a promoter: Mapping with restriction enzyme mediated deletions Conclusion: Light units of luciferase in hepatocytes
20 HSVTK Gancilovir, ATPGancilovir-PO 4 Mammalian TK Gancylovir, ATP toxicity, death Use example: Site-directed recombination Engineered chromosome: WT protein of interest HSVTK lox Replacement plasmid: Mut. protein of interest gancylovir Mut. protein of interest Select recombinants as HSVTK -, gancilovir-resistant Gancyclovir selection AGAINST the presence of enzyme activity CRE recombinase (cassette excnahge) (Ganciclovir itself is not toxic)
21 Footprinting: detects sites on DNA to which protein are bound Naked DNA DNA + DNA-binding protein Population of molecules missing Population of molecules Partial DNase Gel electrophoresis. autoradiography Footprint Further promoter characterization: binding speicificity
22 Note uneven cleavage of naked DNA by DNase
23 (EMSA = electrophoretic mobility shift assay) (shift) (supershift) DNA element U. Arizona Protein-DNA binding: EMSA or gel shift (Even though the hexagon looks like a protein here) competitor
24 (surpershifted complex is not competed by NON- specific probe) (competed only by specific probe) (two molecules of protein bound) Protein DNA complexes migrate more slowly than naked DNA Gel shifts (EMSA Super- shift
25 SELEX Binding to Protein, e.g. sequences consensus by PCR Synthetic, range usually 6 to 40-mers (huge number) Separate using nitrocellulose binding, gel electrophoresis, etc. (re-iterate 3-10 times) (usually a protein) (T7 RNA Pol from an embedded T7Pol promoter ; for protein binding sites Systematic Evolution of Ligands by Exponential Enrichment
26 Binding to protein of interest RT luebeck.de/T.%20Restle/ Bilder/SELEX.jpg Practical capacity: random sequences (random ~21-mer = 4 21 ) Re-adding the T7 promoter sequence on the PCR primer
27 PUM2, a novel murine puf protein, and its consensus RNA-binding site White EK, Moore-Jarrett T, Ruley HE. RNA Dec;7(12): White EKMoore-Jarrett TRuley HE Consensus: Binding site for a “puf “ protein, implicated in mRNA degradation Cod e Intege r Base NameMeanin g Complemen t A1 Adenine AT C2 Cytosine CG G3 Guanine GC T4 Thymine TA U4 Uracil UA R5 (PuRine) G|AG|AY Y6 (PYrimidine) T|CT|CR K7 (Keto) G|TG|TM M8 (AMino) A|CA|CK S9 Strong interaction (3 H bonds) G|CG|CS W10 Weak interaction (2 H bonds) A|TA|TW B11 Not-A (B follows A) G|T|CG|T|CV D12 Not-C (D follows C) G|A|TG|A|TH H13 Not-G (H follows G) A|T|CA|T|CD V14 Not-T (or U) (V follows U) G|A|CG|A|CB N,X15 ANy nucleotide G|A|T|CG|A|T|CN -16 Gap of indeterminate length Gap- Description Nucleic acid degenerate base abbreviations 20-mer
28 Got this far
29 Measuring gene expression via RNA Northern blot RNase protection Primer extension RT-PCR Q-RT-PCR Microarray RNAseq
30 Alternative polyadenylation sites 2 dhfr mRNAs Northern blotting Denaturing gel for true MW (urea, formamide)
31 RNase protection (RPA) Mutant-exon3 Wild type dhfr mRNA
33 Primer extension: map the 5’ end of an mRNA 1313 major start minor start
34 Cap trapping to isolate cDNAs that go to the 5’ end of the mRNA First biotinylate the ribose residues that carry adjacent ring hydroxyls (diols):
35 Full length Truncated Magnetic avidin beads Next: Use an XhoI-tailed adapter-primer to copy the RNA into cDNA Use RNaseI to digest SS RNA. Biotinylated 3’ end cleaved. 5’ incomplete cDNAs lose their cap- biotin. Isolate the surviving capped DS molecules with avidin beads. Get rid of the RNA with RNase A. dG tail. Make second strand with SacI-tailed oligo dC Cut with SacI and XhoI and clone.
36 “Nanostrings” to quantify mRNA levels by single molecule counting 900 nt m13 segments labeled with one of 4 fluorescent dyes. Make a unique color- code, ligate to nt mRNA-specific seq and to a 5’ universal repeat. Can make up to 800 of these. Ligate a universal 3’ repeat to the 3’ end of an mRNA-specific sequence (35-50 nt) Fluorescent RNA: T7 promoted transcription of m13 segment PCR product using amino-allyl-UTP; then conjugate to dye. 4 colors, 7 positions, 3 7 =2100 [diff. neighbors] Strech out via electrophoresis and then anchor far end. Avidin coated surface. B=biotinylated Geiss et al. Nat. Biotech. 26:317, 2008
37 Digital droplet PCR, or digital PCT, dPCR Quantalife (Bio-Rad) PCR in droplets Read + or – in instrument Data λ=average no. of occurrences f= probability of k occurrences For k=0, f 0 =e - λ Observe f, calculate λ Poisson distribution: Aqueous microspheres in water-in-oil emulsion Positive (green, here) microspheres had >= 1 templates. All positives have same intensity, as PCR plateau.
38 Protein-protein interactions Yeast 2-hybrid system Yeast 3-hybrid and 1 hybrid systems Co-immunoprecipitation Pull-downs Far western blots Biacore (surface plasmon resonance, SPR) Fragment complementation
39 Measuring protein-protein interactions in vitro X=one protein Y= another protein Pull-downs: Binding between defined purified proteins, at least one being purified. Tag each protein differently by making the appropriate cDNA clone. Examples: His 6 -X+HA-Y; bind to nickel or cobalt ion column via X, elute (imidazole), Western via anti-HA Ab for Y GST-X + HA-Y; bind to glutathione column, elute (glutathione), Western with anti-HA Ab His 6 -X + 35 S-Y (made in vitro); bind Ni column, elute (imid.), gel + autoradiography. No antibody needed. (HA = flu hemagglutinin) glutathione = gamma-glutamyl-cysteinyl-glycine.
40 Example of a result of a pull-down experiment Antibody used in Western Total protein: no antibody/Western (stained with Coomassie Blue or silver stain) Compare pulled down fraction (eluted) with loaded. Loaded sample usually only a fraction. Also identfy by MW (or mass spec) Western blot Total protein