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Yeast Expression Vector (example)

1. Nov. 8, 2011 1:00 AM. 1. GAPD term’n. LEU2. GAPD prom. Amp r. ori E. Yeast Expression Vector (example). Saccharomyces cerevisiae (baker’s yeast). 2 mu seq features: yeast ori ori E = bacterial ori Amp r = bacterial selection LEU2, e.g. = Leu biosynthesis for yeast selection.

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Yeast Expression Vector (example)

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  1. 1 Nov. 8, 2011 1:00 AM 1 GAPD term’n LEU2 GAPD prom Ampr oriE Yeast Expression Vector (example) Saccharomyces cerevisiae(baker’s yeast) 2 mu seq features: yeast ori oriE = bacterial ori Ampr = bacterial selection LEU2, e.g. = Leu biosynthesisfor yeast selection 2μ = 2 micron plasmid Complementation of an auxotrophy can be used instead of drug-resistance Your favorite gene(Yfg) Auxotrophy = state of a mutant in a biosynthetic pathway resulting in a requirement for a nutrient For growth in E. coli GAPD = the enzyme glyceraldehyde-3 phosphate dehydrogenase

  2. 2 Vector DNA t p gfY Genomic DNA Genomic DNA HIS4 mutation- Yeast - genomic integration via homologous recombination HIS4 t p Yfg FunctionalHIS4 gene DefectiveHIS4 gene

  3. 3 Vector DNA Yfg Genomic DNA AOX1 gene (~ 30% of total protein) Genomic DNA Yfg 3’AOX1 AOX1p AOX1t HIS4 Double recombination Yeast (integration in Pichia pastoris) HIS4 P. pastoris-tight control-methanol induced (AOX1)-large scale production (gram quantities) AOX1t AOX1p 3’AOX1 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. 1999. 400:464-8) 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) Cos Monkey cells with SV40 replication proteins (-> high transgene copies) 3T3 Mouse or human exhibiting ~regulated (normal-like) growth + various others, many differentiated to different degrees, e.g.: BHK Baby hamster kidney HepG2 Human hepatoma GH3 Rat pituitary cells PC12 Mouse neuronal-like tumor cells MCF7 Human breast cancer HT1080 Human fibroblastic cells with near diploid karyotype IPS induced pluripotent stem cells and: Common in industry for production: NS1 mAbs Mouse plasma cell tumor cells Vero vaccines African greem monkey cells CHO mAbs, other therapeutic proteins Chinese hamster ovary cells PER6 mAbs, other therapeutic proteins Human retinal cells

  6. Mammalian cell expression Generalized gene structure for mammalian expression: polyA site intron Mam.prom. 3’UTR cDNA gene 5’UTR Intron is optional but a good idea

  7. 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) Actin EIF2alpha (EIF = eukaryotic initiation [of translation] factor) Engineered inducible / repressible:tet, ecdysone, glucocorticoid (tet = tetracycline) Popular mammalian cell promoters

  8. Engineered regulated expression: Tetracycline-reponsive promoters Tet-OFF (add tet  shut off) Tet-OFF VP16 transcriptionactivation domain tetRdomain tTA = tet activator fusion protein: tetR = tet repressor (original role) active No tet.Binds tet operator (multiple copies)(if tet not also bound) VP16 transcriptionactivation domain tetRdomain Tet-OFF Allosteric change in conformation Tetracycline (tet), or,better, doxicyclin (dox) not active tTA gene must be in cell (permanent transfection, integrated): polyA site CMV prom. tTA cDNA (Bujold et al.)

  9. tetRdomain VP16 tc’nact’n domain not active little transcripton (2%?, bkgd) Doxicyclin present: polyA site MIN. CMV prom. your favorite gene polyA site polyA site your favorite gene your favorite gene No doxicyclin: VP16 tc’nact’n domain tetRdomain active Plenty of transcripton RNA po l MIN. CMV prom. Tet-OFF, cont. MIN. CMV prom. Mutliple tet operator elements

  10. Tet-ON Tetracycline-reponsive promoters Tet-ON (add tet  turn on gene tetRdomain VP16 tc’nact’n domain not active Different fusion protein: Does NOT bind tet operator(if tet not bound) tetRdomain VP16 tc’nact’n domain active Tetracycline (tet), or,better, doxicyclin (dox) polyA site Full CMV prom. tTA cDNA Must be in cell (permanent transfection, integrated): commercially available (293, CHO) or do-it-yourself

  11. polyA site polyA site polyA site your favorite gene your favorite gene your favorite gene Tet-ON MIN. CMV prom. Mutliple tet operator elements tetRdomain VP16 tc’nact’n domain not active little transcription (bkgd.) Doxicyclin absent: MIN. CMV prom. Add dox: active tetRdomain VP16 tc’nact’n domain doxicyclin active Plenty of transcripton (> 50X) RNA pol II MIN. CMV prom.

  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 Core transcriptional elements TF… transcription factor TBP: TATA binding protein TAF: TBP associated protein BRE: TFIIB response element INR: transcription initiator element DPE: downstream promoter element -28 -35 GGGCGCC; CCACGCC YYAN(TA)YY (AG)G(AT)(CT)(GAC) TATA(AT)AA(GA) Y = C or T (pyrimidine) The transcription complex either recruits RNA Pol II or activates a bound RNA Pol II For review see Smale and Katonga, Ann. Rev. Biochem. 72: 449-479 (2003)

  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 Space for res. enz. to bind Reportergene PCR Transfect

  16. 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 (G418R) (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) Popular reporters to study promoter/enhancers FACS = fluorescence-activated cell sorter

  17. Testing for a cell-specific promoter: chloramphenicol acetyl transferase (CAT) reporter assay (www.biochem.arizona.edu/classes/bioc471/pages/Lecture15/Lecture15.html) CAT cDNA is from a prokaryotic source. CAT is not found in mammalian cells. Therefore low backgrounds diacetylated B A Thin layer chromatography (TLC) 14C-chloramphenicol monoacetylated unacetylated Positive control Negative control

  18. 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. Reporter enzyme substrates for different purposes Substrates for beta-galactosidase, for example:

  19. Light units of luciferase in hepatocytes Mapping transcriptional elements upstream of a promoter: Mapping with restrictionenzyme mediated deletions Conclusion:

  20. CRE recombinase (cassette excnahge) Mut. protein of interest Gancyclovir selection AGAINST the presence of enzyme activity HSVTK Gancilovir, ATP Gancilovir-PO4 toxicity, death Mammalian TK Gancylovir, ATP (Ganciclovir itself is not toxic) Use example: Site-directed recombination Engineered chromosome: lox lox WT protein of interest HSVTK Replacement plasmid: gancylovir Mut. protein of interest Select recombinants as HSVTK-, gancilovir-resistant

  21. Further promoter characterization: binding speicificity Footprinting: detects sites on DNA to which protein are bound DNA + DNA-binding protein Naked DNA Population of molecules Partial DNase Population of molecules missing Gel electrophoresis.autoradiography Footprint

  22. Note uneven cleavage of naked DNA by DNase

  23. Protein-DNA binding: EMSA or gel shift (EMSA = electrophoretic mobility shift assay) 1 2 3 4 5 competitor (supershift) (shift) DNA element (Even though the hexagon looks like a protein here) U. Arizona

  24. Gel shifts (EMSA (surpershifted complex is not competed by NON-specific probe) Protein DNA complexes migrate more slowly than naked DNA (competed only by specific probe) Super- shift (two molecules of protein bound)

  25. SELEX for protein binding sites Systematic Evolution of Ligands by Exponential Enrichment (T7 RNA Pol from an embedded T7Pol promoter (huge number) Synthetic, range usually 6 to 40-mers (usually a protein) ; by PCR (re-iterate 3-10 times) Binding to Protein, e.g. Separate using nitrocellulose binding, gel electrophoresis, etc. sequences  consensus

  26. Practical capacity: 1014 random sequences (random ~21-mer = 421) Re-adding the T7 promoter sequence on the PCR primer Binding to protein of interest http://www.molmed.uni-luebeck.de/T.%20Restle/Bilder/SELEX.jpg RT

  27. Binding site for a “puf “ protein, implicated in mRNA degradation PUM2, a novel murine puf protein, and its consensus RNA-binding siteWhite EK, Moore-Jarrett T, Ruley HE. RNA. 2001 Dec;7(12):1855-66. 20-mer Nucleic acid degenerate base abbreviations Consensus: Description

  28. Got this far

  29. Northern blot RNase protection Primer extension RT-PCR Q-RT-PCR Microarray RNAseq Measuring gene expression via RNA

  30. Northern blotting Denaturing gel for true MW (urea, formamide) Alternative polyadenylation sites  2 dhfr mRNAs http://www.gene-quantification.de/mrna.html#northern

  31. RNase protection (RPA) dhfr mRNA Wild type Mutant-exon3 1-2-3-4-5-6 1-2-4-5-6

  32. Primer extension: map the 5’ end of an mRNA 1 3 minor start major start

  33. 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):

  34. 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. Full length Truncated Magnetic avidin beads

  35. “Nanostrings” to quantify mRNA levels by single molecule counting Geiss et al. Nat. Biotech. 26:317, 2008 900 nt m13 segments labeled with one of 4fluorescent dyes. Make a unique color-code, ligate to 30-50- nt mRNA-specific seq and to a 5’ universal repeat. Can make up to 800 of these. Strech out via electrophoresis and then anchor far end. Ligate a universal 3’ repeat to the 3’ end of an mRNA-specific sequence (35-50 nt) Avidin coated surface.B=biotinylated Fluorescent RNA: T7 promoted transcription of m13 segment PCR product using amino-allyl-UTP; then conjugate to dye. 4 colors, 7 positions, 37=2100 [diff. neighbors]

  36. λ=average no. of occurrences f= probability of k occurrences For k=0, f0=e-λ Observe f, calculateλ Digital droplet PCR, or digital PCT, dPCR Quantalife (Bio-Rad) Poisson distribution: Aqueous microspheres in water-in-oil emulsion PCR in droplets Read + or – in instrument Data Positive (green, here) microspheres had >= 1 templates. All positives have same intensity, as PCR  plateau.

  37. 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

  38. 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: His6-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 His6-X + 35S-Y (made in vitro); bind Ni column, elute (imid.),  gel + autoradiography. No antibody needed. (HA = flu hemagglutinin) glutathione = gamma-glutamyl-cysteinyl-glycine.

  39. Example of a result of a pull-down experiment Western blot Total protein Also identfy by MW (or mass spec) Total protein: no antibody/Western (stained with Coomassie Blue or silver stain) Antibody used in Western Compare pulled down fraction (eluted)with loaded. Loaded sample usually only a fraction.

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