Presentation on theme: "Yeast Expression Vector (example)"— Presentation transcript:
1Yeast Expression Vector (example) 1Nov. 8, :00 AM1Yeast Expression Vector (example)Saccharomyces cerevisiae (baker’s yeast)2 mu seq features:yeast orioriE = bacterial oriAmpr = bacterial selectionLEU2, e.g. = Leu biosynthesis for yeast selection2μ = 2 micron plasmidGAPD term’nGAPD promLEU2AmprComplementation of an auxotrophy can be used instead of drug-resistanceoriEYour favorite gene (Yfg)Auxotrophy = state of a mutant in a biosynthetic pathway resulting in a requirement for a nutrientFor growth in E. coliGAPD = the enzyme glyceraldehyde-3 phosphate dehydrogenase
3Double recombination Yeast (integration in Pichia pastoris) 3Double recombination Yeast (integration in Pichia pastoris)P. pastoris -tight control -methanol induced (AOX1) -large scale production (gram quantities)HIS4Vector DNAAOX1tYfgAOX1p3’AOX1AOX1 gene (~ 30% of total protein)Genomic DNAAlcohol oxidase geneAOX1pYfgAOX1tHIS43’AOX1Genomic DNA
4Primary cells cultured with a limited lifetime. E.g., MEF = mouse embryonic fibroblasts, HDF = Human diploid fibroblastsMammalian 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 survivorsHuman 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)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).
5Expression 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 kidneyHepG2 Human hepatomaGH3 Rat pituitary cellsPC12 Mouse neuronal-like tumor cellsMCF7 Human breast cancerHT1080 Human fibroblastic cells with near diploid karyotypeIPS induced pluripotent stem cellsand:Common in industry for production:NS1 mAbs Mouse plasma cell tumor cellsVero vaccines African greem monkey cellsCHO mAbs, other therapeutic proteins Chinese hamster ovary cellsPER6 mAbs, other therapeutic proteins Human retinal cells
6Mammalian cell expression Generalized gene structure for mammalian expression:polyA siteMam.prom.cDNA geneintron3’UTR5’UTRIntron is optional but a good idea
8Engineered regulated expression: Tetracycline-reponsive promoters Tet-OFF (add tet shut off)Tet-OFFtetR domainVP16 transcription activation domaintTA = tet activator fusion protein:tetR = tet repressor (original role)activeNo tet. Binds tet operator (multiple copies) (if tet not also bound)VP16 transcription activation domaintetR domainTet-OFFAllosteric change in conformationTetracycline (tet), or, better, doxicyclin (dox)not activetTA gene must be in cell (permanent transfection, integrated):polyA siteCMV prom.tTA cDNA(Bujold et al.)
9Tet-OFF, cont. polyA site your favorite gene No doxicyclin: active MIN. CMV prom.Mutliple tet operator elementsMIN. CMV prom.your favorite genepolyA siteactivePlenty of transcriptonNo doxicyclin:tetR domainVP16 tc’n act’n domainRNA po lMIN. CMV prom.your favorite genepolyA sitetetR domainVP16 tc’n act’n domainnot activelittle transcripton (2%?, bkgd)Doxicyclin present:
10Tetracycline-reponsive promoters Tet-ON (add tet turn on gene tetR domainVP16 tc’n act’n domainDifferent fusion protein: Does NOT bind tet operator (if tet not bound)not activetetR domainVP16 tc’n act’n domainactiveTetracycline (tet), or, better, doxicyclin (dox)polyA siteFull CMV prom.tTA cDNAMust be in cell (permanent transfection, integrated):commercially available (293, CHO) or do-it-yourself
11Tet-ON polyA site your favorite gene Mutliple tet operator elements MIN. CMV prom.Mutliple tet operator elementstetR domainVP16 tc’n act’n domainnot active little transcription (bkgd.)Doxicyclin absent:your favorite genepolyA siteMIN. CMV prom.Add dox:activetetR domainVP16 tc’n act’n domaindoxicyclinactivePlenty of transcripton (> 50X)your favorite genepolyA siteRNA pol IIMIN. CMV prom.
12Biotechnology 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:
13General model for transcriptional regulation in higher eukaryotes Core transcriptional elementsTF… transcription factorTBP: TATA binding proteinTAF: TBP associated proteinBRE: TFIIB response elementINR: transcription initiator elementDPE: downstream promoter elementStrachan p. 175 fig. 8.4-35-28GGGCGCC;CCACGCCTATA(AT)AA(GA)YYAN(TA)YY(AG)G(AT)(CT)(GAC)Y = C or T (pyrimidine)The transcription complex either recruits RNA Pol II or activates a bound RNA Pol IIFor review see Smale and Katonga, Ann. Rev. Biochem. 72: (2003)
14Many transcriptional enhancer elements often lie upstream of promoters, allowing for many combinations of TF bindingStrachan p. 175 fig. 8.5
15Space for res. enz. to bind Put a DNA regulatory region upstream of a reporter gene to analyze its elementsSpace for res. enz. to bindReporter genePCRStrachan p. 482Transfect
16Popular reporters to study promoter/enhancers Beta-galactosidase (β-gal) – detection by several different assaysChloramphenicol acetyl transferase (CAT) – detection, sensitive radioactive assayLuciferase (firefly, Renilla [jellyfish]) – detection, easy dual, sensitive luminescent assayGreen fluorescent protein (GFP, BFP, YFP)) – cytological, visible in living cells, fusion proteins, FACSNeomycin phosphotransferase (neo)–selectable drug resistance (G418R)(similarly: resistance to hygromycin, puromycin, histidinol, zeocin)Dihydrofolate reductase (DHFR) – selectable in dhfr- cells, amplifiable, fusion proteins workSuicide selection: Herpes simplex virus thymidine kinase (HSVTK)FACS = fluorescence-activated cell sorter
17Testing 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 foundin mammalian cells.Therefore low backgroundsdiacetylatedABThin layerchromatography (TLC)14C-chloramphenicolmonoacetylatedunacetylatedPositive controlNegative control
18Reporter enzyme substrates for different purposes Substrates for beta-galactosidase, for example: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 detectionUmbelliferyl–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.
19Light units of luciferase in hepatocytes Mapping transcriptional elements upstream of a promoter:Mapping with restriction enzyme mediated deletionsMiesfeld p. 104Conclusion:
20Gancyclovir selection AGAINST the presence of enzyme activity HSVTKGancilovir, ATPGancilovir-PO4toxicity, deathMammalian TKGancylovir, ATP(Ganciclovir itself is not toxic)Use example: Site-directed recombinationEngineered chromosome:loxloxWT protein of interestHSVTKCRE recombinase(cassette excnahge)Replacement plasmid:Mut. protein of interestgancylovirMut. protein of interestSelect recombinants as HSVTK-, gancilovir-resistant
21Gel electrophoresis. autoradiography Further promoter characterization: binding speicificityFootprinting: detects sites on DNA to which protein are boundNaked DNADNA + DNA-binding proteinPopulation of moleculesPartial DNaseStrachan p. 484Population of moleculesmissingGel electrophoresis. autoradiographyFootprint
22Note uneven cleavage of naked DNA by DNase Strachan p. 484
23Protein-DNA binding: EMSA or gel shift (EMSA = electrophoretic mobility shift assay)competitorMiesfeld p. 108(supershift)(shift)DNA element(Even though the hexagon looks like a protein here)U. Arizona
24(surpershifted complex is not competed by NON-specific probe) 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)Miesfeld p. 108
25SELEX for protein binding sites Systematic Evolution of Ligands by Exponential Enrichment(T7 RNA Pol from an embedded T7Pol promoterSynthetic, range usually 6 to 40-mers(huge number)Selex(usually a protein);by PCR(re-iterate 3-10 times)Binding to Protein,e.g.Separate using nitrocellulose binding, gel electrophoresis, etc.sequences consensus
26Binding to protein of interest Practical capacity:1014 random sequences(random ~21-mer = 421)Re-adding the T7 promoter sequence on the PCR primerBinding to protein of interestRT
27Binding site for a “puf “ protein, implicated in mRNA degradation PUM2, a novel murine puf protein, and its consensus RNA-binding site White EK, Moore-Jarrett T, Ruley HE. RNA Dec;7(12):20-merNucleic acid degenerate base abbreviationsCodeIntegerBase NameMeaningComplementA1AdenineTC2CytosineG3Guanine4ThymineUUracilR5(PuRine)G|AY6(PYrimidine)T|CK7(Keto)G|TM8(AMino)A|CS9Strong interaction (3 H bonds)G|CW10Weak interaction (2 H bonds)A|TB11Not-A (B follows A)G|T|CVD12Not-C (D follows C)G|A|TH13Not-G (H follows G)A|T|C14Not-T (or U) (V follows U)G|A|CN,X15ANy nucleotideG|A|T|CN-16Gap of indeterminate lengthGapConsensus:Description
33Primer extension: map the 5’ end of an mRNA 13minor startmajor start
34Cap trapping to isolate cDNAs that go to the 5’ end of the mRNA First biotinylate the ribose residues that carry adjacent ring hydroxyls (diols):
35Use an XhoI-tailed adapter-primer to copy the RNA into cDNA Next:Use an XhoI-tailed adapter-primer to copy the RNA into cDNAUse 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 dCCut with SacI and XhoI and clone.Full lengthTruncatedMagnetic avidin beads
36“Nanostrings” to quantify mRNA levels by single molecule counting Geiss et al. Nat. Biotech. 26:317, 2008900 nt m13 segmentslabeled 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.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=biotinylatedFluorescent 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]
37Digital droplet PCR, or digital PCT, dPCR Quantalife (Bio-Rad) λ=average no. of occurrencesf= probability of k occurrencesFor k=0, f0=e-λObserve f, calculate λPoisson distribution:Aqueous microspheres in water-in-oil emulsionPCR in dropletsRead + or – in instrumentDataPositive (green, here) microspheres had >= 1 templates. All positives have same intensity, as PCR plateau.
38Protein-protein interactions Yeast 2-hybrid systemYeast 3-hybrid and 1 hybrid systemsCo-immunoprecipitationPull-downsFar western blotsBiacore (surface plasmon resonance, SPR)Fragment complementation
39Measuring protein-protein interactions in vitro X=one protein Y= another proteinPull-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 YGST-X + HA-Y; bind to glutathione column, elute (glutathione), Western with anti-HA AbHis6-X S-Y (made in vitro); bind Ni column, elute (imid.), gel + autoradiography No antibody needed.(HA = flu hemagglutinin) glutathione = gamma-glutamyl-cysteinyl-glycine.
40Example of a result of a pull-down experiment Western blotTotal proteinAlso identfy by MW(or mass spec)Total protein: no antibody/Western(stained with Coomassie Blue or silver stain)Antibody used in WesternCompare pulled down fraction (eluted) with loaded. Loaded sample usually only a fraction.