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Yeast Expression Vector (example)

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1 Yeast Expression Vector (example)
1 Nov. 8, :00 AM 1 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 biosynthesis for yeast selection 2μ = 2 micron plasmid GAPD term’n GAPD prom LEU2 Ampr Complementation of an auxotrophy can be used instead of drug-resistance oriE 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 Yeast - genomic integration via homologous recombination
2 Yeast - genomic integration via homologous recombination gfY p t Vector DNA HIS4 Genomic DNA HIS4 mutation- Genomic DNA p t Yfg Functional HIS4 gene Defective HIS4 gene

3 Double recombination Yeast (integration in Pichia pastoris)
3 Double recombination Yeast (integration in Pichia pastoris) P. pastoris -tight control -methanol induced (AOX1) -large scale production (gram quantities) HIS4 Vector DNA AOX1t Yfg AOX1p 3’AOX1 AOX1 gene (~ 30% of total protein) Genomic DNA Alcohol oxidase gene AOX1p Yfg AOX1t HIS4 3’AOX1 Genomic DNA

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) 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 Mam.prom. cDNA gene intron 3’UTR 5’UTR Intron is optional but a good idea

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

8 Engineered regulated expression: Tetracycline-reponsive promoters
Tet-OFF (add tet  shut off) Tet-OFF tetR domain VP16 transcription activation domain tTA = tet activator fusion protein: tetR = tet repressor (original role) active No tet. Binds tet operator (multiple copies) (if tet not also bound) VP16 transcription activation domain tetR domain 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 Tet-OFF, cont. polyA site your favorite gene No doxicyclin: active
MIN. CMV prom. Mutliple tet operator elements 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 MIN. CMV prom. your favorite gene polyA site tetR domain VP16 tc’n act’n domain not active little transcripton (2%?, bkgd) Doxicyclin present:

10 Tetracycline-reponsive promoters Tet-ON (add tet  turn on gene
tetR domain VP16 tc’n act’n domain Different fusion protein: Does NOT bind tet operator (if tet not bound) not active tetR domain VP16 tc’n act’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 Tet-ON polyA site your favorite gene Mutliple tet operator elements
MIN. CMV prom. Mutliple tet operator elements tetR domain VP16 tc’n act’n domain not active little transcription (bkgd.) Doxicyclin absent: your favorite gene polyA site MIN. CMV prom. Add dox: active tetR domain VP16 tc’n act’n domain doxicyclin active Plenty of transcripton (> 50X) your favorite gene polyA site 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 Strachan p. 175 fig. 8.4 -35 -28 GGGCGCC; CCACGCC TATA(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 II For review see Smale and Katonga, Ann. Rev. Biochem. 72: (2003)

14 Many transcriptional enhancer elements often lie upstream of promoters, allowing for many combinations of TF binding Strachan p. 175 fig. 8.5

15 Space for res. enz. to bind
Put a DNA regulatory region upstream of a reporter gene to analyze its elements Space for res. enz. to bind Reporter gene PCR Strachan p. 482 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 (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) FACS = fluorescence-activated cell sorter

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

18 Reporter 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 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.

19 Light units of luciferase in hepatocytes
Mapping transcriptional elements upstream of a promoter: Mapping with restriction enzyme mediated deletions Miesfeld p. 104 Conclusion:

20 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 CRE recombinase (cassette excnahge) Replacement plasmid: Mut. protein of interest gancylovir Mut. protein of interest Select recombinants as HSVTK-, gancilovir-resistant

21 Gel electrophoresis. autoradiography
Further promoter characterization: binding speicificity Footprinting: detects sites on DNA to which protein are bound Naked DNA DNA + DNA-binding protein Population of molecules Partial DNase Strachan p. 484 Population of molecules missing Gel electrophoresis. autoradiography Footprint

22 Note uneven cleavage of naked DNA by DNase
Strachan p. 484

23 Protein-DNA binding: EMSA or gel shift
(EMSA = electrophoretic mobility shift assay) competitor Miesfeld 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

25 SELEX for protein binding sites
Systematic Evolution of Ligands by Exponential Enrichment (T7 RNA Pol from an embedded T7Pol promoter Synthetic, 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

26 Binding to protein of interest
Practical capacity: 1014 random sequences (random ~21-mer = 421) Re-adding the T7 promoter sequence on the PCR primer Binding to protein of interest RT

27 Binding 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-mer Nucleic acid degenerate base abbreviations Code Integer Base Name Meaning Complement A 1 Adenine T C 2 Cytosine G 3 Guanine 4 Thymine U Uracil R 5 (PuRine) G|A Y 6 (PYrimidine) T|C K 7 (Keto) G|T M 8 (AMino) A|C S 9 Strong interaction (3 H bonds) G|C W 10 Weak interaction (2 H bonds) A|T B 11 Not-A (B follows A) G|T|C V D 12 Not-C (D follows C) G|A|T H 13 Not-G (H follows G) A|T|C 14 Not-T (or U) (V follows U) G|A|C N,X 15 ANy nucleotide G|A|T|C N - 16 Gap of indeterminate length Gap Consensus: Description

28 Got this far

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

30 Denaturing gel for true MW (urea, formamide)
Northern blotting Denaturing gel for true MW (urea, formamide) Alternative polyadenylation sites  2 dhfr mRNAs

31 RNase protection (RPA)
dhfr mRNA Mutant-exon3 Wild type


33 Primer extension: map the 5’ end of an mRNA
1 3 minor start major 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 Use an XhoI-tailed adapter-primer to copy the RNA into cDNA
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

36 “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 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=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]

37 Digital droplet PCR, or digital PCT, dPCR Quantalife (Bio-Rad)
λ=average no. of occurrences f= probability of k occurrences For k=0, f0=e-λ Observe f, calculate λ 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.

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: 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 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
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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