1. BioSci 203 lecture 21 page 1 © copyright Bruce Blumberg 2001. All rights reserved Bio Sci 203 Lecture 21 - cDNA library screening &sequence characterization Bruce Blumberg (blumberg@uci.edu) –office - 4203 McGaugh Hall –824-8573 –lab 5427 (x46873), 5305 (x43116) –office hours Wednesday 1-2. This week –Finish up cDNA identification –Protein protein binding assays –Characterization of Selected DNA Sequences DNA sequence analysis –mRNA Analysis Techniques to detect and quantitate mRNA –Northern –RNase protection –RT-PCR –in-situ hybridization –Transcript mapping

2. BioSci 203 lecture 21 page 2 © copyright Bruce Blumberg 2001. All rights reserved How to identify your gene of interest (contd) A DNA element required for expression of an interesting gene? –How to identify what factors bind to putative elements? examine the sequence –does it contain known binding sites? –if yes, do such proteins bind to the isolated element in gel-shift experiments? do the elements bind proteins from nuclear extracts? –gel shift (EMSA) experiments clone the elements into reporters with minimal promoters. –do these constructs recapitulate activity? –What does the sequence tell you about the binding protein? AGGTCATGACCT

3. BioSci 203 lecture 21 page 3 © copyright Bruce Blumberg 2001. All rights reserved How to identify your gene of interest (contd) Biochemical purification of binding proteins –tedious, considerable biochemical skill required –two basic approaches fractionate nuclear extracts chromatographically and test fractions for ability to bind the element DNA-affinity chromatography –multimerize the element and bind to a resin –pass nuclear extracts across column and purify specific binding proteins –protein microsequencing –predict DNA sequence from amino acid sequence look in the database prepare oligonucleotides and screen library –advantages gold standard if you can purify proteins, this will always work –not so many good protein biochemists works for dimeric proteins and complexes –disadvantages slow, tedious need good protein sequencing facility biochemical expertise required expense of preparing preparative quantities of nuclear extracts

4. BioSci 203 lecture 21 page 4 © copyright Bruce Blumberg 2001. All rights reserved How to identify your gene of interest (contd) Molecular biological approaches to identifying binding proteins –oligonucleotide screening of expression libraries (Singh screening) multimerize oligonucleotide and label with 32 P screen expression library to identify binding proteins advantages –straightforward –much less biochemical expertise required than biochemical purification –relatively fast disadvantages –can’t detect binding if multiple partners are required –fair amount of “touch” required

5. BioSci 203 lecture 21 page 5 © copyright Bruce Blumberg 2001. All rights reserved How to identify your gene of interest (contd)) Molecular biological approaches to identifying binding proteins –expression cloning (sib screening) clone element of interest (or promoter) into a suitable reporter construct (e.g. luciferase) transfect (or inject, or infect, etc) pools (~10,000 cDNAs each) of cDNA expression libraries and assay for reporter gene retest positive pools in smaller aliquots (~1000) repeat until a pure cDNA is found –advantages functional approach presumably using the appropriate cell type so modifications occur possibility to detect dimers with endogenous proteins –disadvantages VERY TEDIOUS very slow, much duplication in pools, extensive rescreening is required could be expensive

6. BioSci 203 lecture 21 page 6 © copyright Bruce Blumberg 2001. All rights reserved How to identify your gene of interest (contd) Molecular biological approaches to identifying binding proteins –in vitro expression cloning (IVEC) transcribe and translate cDNA libraries in vitro into small pools of proteins (~100) EMSA to test protein pools for element binding unpool cDNAs and retest advantages –functional approach –smaller pools increase sensitivity disadvantages –can’t detect dimers –very expensive (TNT lysate) –considerable rescreening still required –tedious, countless DNA minipreps required

7. BioSci 203 lecture 21 page 7 © copyright Bruce Blumberg 2001. All rights reserved Identification of binding proteins (contd) Molecular biological approaches to identifying binding proteins (contd) –hybrid screening system 1 begin with cDNA libraries in 384-well plates, 1 cDNA per well pool cDNAs using robotic workstation prepare DNA with robotic workstation transcribe and translate protein in vitro test for ability to bind DNA element using sensitive, high-throughput assay –fluorescence –radioactive assay retest components of positive pools advantages –very fast, only two steps required, ~ 2 weeks –little work required disadvantages –expense of robotics –won’t detect dimers (unless 1 partner known) –expense of reagents (TNT, radionuclides, fluorescent labels

8. BioSci 203 lecture 21 page 8 © copyright Bruce Blumberg 2001. All rights reserved Identification of binding proteins (contd) advantages –very fast, only two steps required, ~ 2 weeks –little work required disadvantages –expense of robotics –won’t detect dimers (unless 1 partner known) –expense of reagents (TNT, radionuclides, fluorescent labels

9. BioSci 203 lecture 21 page 9 © copyright Bruce Blumberg 2001. All rights reserved Identification of binding proteins (contd) –hybrid screening system 2 prepare reporter cell line with element or promoter driving reporter gene (e.g. luciferase) prepare cDNA pools as in system 1 use robotic workstation to transfect cDNA libraries into reporter cells assay for reporter gene advantages –very fast –truly functional approach –use of cells allows modifications –can detect dimers if one partner is already present in cell disadvantages –expense of equipment

10. BioSci 203 lecture 21 page 10 © copyright Bruce Blumberg 2001. All rights reserved How to identify your gene of interest (contd) Molecular approaches (contd) –yeast one-hybrid assay clone element of interest into a reporter construct and make stable yeast strain transfect aliquots of cDNA expression libraries that have fragments of DNA fused to yeast activator if the fusion protein binds to your element then the reporter gene will be activated advantages –somewhat of a functional approach –eukaryotic milieu allows some protein modification disadvantages –slow, tedious purification of positives –can’t readily detect heterodimeric proteins –sensitivity is not so great –usual yeast false positives

11. BioSci 203 lecture 21 page 11 © copyright Bruce Blumberg 2001. All rights reserved How to identify your gene of interest (contd) You have one protein and want to identify proteins that interact with it –some sort of interaction screen is indicated straight biochemistry phage display two hybrid in vitro expression cloning biochemical approach –purify cellular proteins that interact with your protein co-immunoprecipitation affinity chromatography biochemical fractionation –pure protein(s) are microsequenced if not in database then make oligonucleotides and screen cDNA library from appropriate tissues –advantage functional approach stringency can be manipulated can identify multimeric proteins or complexes will work if you can purify proteins –disadvantages much skill required low throughput considerable optimization required

12. BioSci 203 lecture 21 page 12 © copyright Bruce Blumberg 2001. All rights reserved How to identify your gene of interest (contd) Phage display screening (a.k.a. panning) –requires a library that expresses inserts as fusion proteins with a phage capsid protein most are M13 based some lambda phages used –prepare target protein as affinity matrix or as radiolabeled probe –test for interaction with library members if using affinity matrix you purify phages from a mixture

13. BioSci 203 lecture 21 page 13 © copyright Bruce Blumberg 2001. All rights reserved How to identify your gene of interest (contd) –test for interaction with library members (contd) if labeling protein one plates fusion protein library and probes with the protein –called receptor panning based on similarity with panning for gold –advantages stringency can be manipulated if the affinity matrix approach works the cloning could go rapidly –disadvantages Fusion proteins bias the screen against full- length cDNAs Multiple attempts required to optimize binding Limited targets possible may not work for heterodimers unlikely to work for complexes panning can take many months for each screen

14. BioSci 203 lecture 21 page 14 © copyright Bruce Blumberg 2001. All rights reserved How to identify your gene of interest (contd) Two hybrid screening –originally used in yeast, now other systems possible –prepare bait - target protein fused to DBD (GAL4) usual stable cell line is commonly used –prepare library as fusion proteins with a known activation domain

15. BioSci 203 lecture 21 page 15 © copyright Bruce Blumberg 2001. All rights reserved How to identify your gene of interest (contd) Two hybrid screening (contd) –approach transfect library into cells and either select for survival or activation of reporter gene purify and characterize positive clones –advantages seems simple and inexpensive on its face –in materials functional assay –disadvantages fusion proteins bias the screen against full-length cDNAs. Binding parameters not manipulable bait must not have activation function Difficult or impossible to detect interactions between proteins and complexes. Doesn’t work for secreted proteins Many months to screen –savings in materials are eaten up by salaries –avg grad student costs $30k/year –avg postdoc or tech costs $40k/year MANY false positives

16. BioSci 203 lecture 21 page 16 © copyright Bruce Blumberg 2001. All rights reserved How to identify your gene of interest (contd) In vitro interaction screening –based on in vitro expression cloning (IVEC) transcribe and translate cDNA libraries in vitro into small pools of proteins (~100) test these proteins for their ability to interact with your protein of interest –EMSA –co-ip –FRET –SPA –advantages functional approach smaller pools increase sensitivity automated variant allows diversity of targets –proteins, protein complexes, nucleic acids, protein/nucleic acid complexes, small molecule drugs –very fast –disadvantages can’t detect heterodimers unless 1 partner known expensive consumables (but cheap salaries) –typical screen will cost $10-15K expense of automation

17. BioSci 203 lecture 21 page 17 © copyright Bruce Blumberg 2001. All rights reserved Current generation binding assays scintillation proximity assay –Target is bound to solid phase - bead or plate –radioactive protein or ligand is added and allowed to reach equilibrium 35 S, 125 I, 3 H work best –radioactive decay is quenched in solution, only detected when in “proximity” of the solid phase, e.g. when bound to target –applications ligand-receptor binding with 3 H small molecules protein:protein interaction protein:DNA

18. BioSci 203 lecture 21 page 18 © copyright Bruce Blumberg 2001. All rights reserved Current generation binding assays SPA (contd –advantages homogeneous, equilibrium assay –washing is not necessary! Can detect weak interactions, insensitive to high off rate large dynamic range –disadvantages radioactive equipment in vitro only

19. BioSci 203 lecture 21 page 19 © copyright Bruce Blumberg 2001. All rights reserved Current generation binding assays (contd) FRET - fluorescent resonance energy transfer –based on the transfer of energy from one fluor to another that is not normally excited at that wavelength –Many types of fluorescent moieties possible rare earth metals europium cryptate fluorescent proteins –GFP and variants –allophycocyanin Tryptophan residues in proteins –application very commonly used for protein:protein interaction screening in industry FRET microscopy can be used to prove interactions between proteins within single cells –Roger Tsien

20. BioSci 203 lecture 21 page 20 © copyright Bruce Blumberg 2001. All rights reserved Current generation binding assays (contd) FRET (contd) –advantages can be very sensitive may be inexpensive or not depending on materials non-radioactive equilibrium assay single cell protein:protein interactions possible time resolved assays possible –disadvantage poor dynamic range - 2-3 fold difference full scale must prepare labeled proteins or ligands tunable fluorometer required (we have one here)

21. BioSci 203 lecture 21 page 21 © copyright Bruce Blumberg 2001. All rights reserved Current generation binding assays (contd) AlphaScreen - amplified luminescent resonance proximity –Bind proteins to two types of beads donor bead accepts laser light at 680 nm and emits singlet oxygen acceptor bead receives singlet oxygen and emits light at 520-620 –principle is that singlet oxygen can only diffuse one bead diameter before decaying only closely association between donor and acceptor gives a signal –applications receptor:ligand binding protein:protein binding discovery of peptide ligands for proteins

22. BioSci 203 lecture 21 page 22 © copyright Bruce Blumberg 2001. All rights reserved Current generation binding assays (contd) AlphaScreen (contd) –advantages very sensitive, equilibrium assay very fast –disadvantages requires instrument ~$100K cost of beads must bind proteins to beads single source for beads and instrument completely in vitro assay

23. BioSci 203 lecture 21 page 23 © copyright Bruce Blumberg 2001. All rights reserved Current generation binding assays (contd) BRET2 (Bioluminescence Resonance Energy Transfer) –based completely on bioluminescent reaction from Renilla reniformis can make lots of money by copying nature! –Renilla luciferase emits blue light in presence of its substrate coelenterazine –If GFP is nearby, it accepts this blue light and emits green light make two fusion proteins, one to rluc the other to GFP mix everything together if proteins interact then green light is detected if not, only blue light

24. BioSci 203 lecture 21 page 24 © copyright Bruce Blumberg 2001. All rights reserved Current generation binding assays (contd) BRET2 (contd) –applications protein protein interactions in solution protein:protein interactions within a single cell –advantages fairly sensitive no laser required to excite –minimal equipment required works in living cells (substrate is permeable) –disadvantage need to make fusion proteins single source for reagents

25. BioSci 203 lecture 21 page 25 © copyright Bruce Blumberg 2001. All rights reserved Current generation binding assays (contd) Eletrochemiluminescent assays –Origen system IGEN –based on a molecule that emits light when stimulated in an electrical field ruthenium derivative –capture a molecule with magnetic beads, if binding occurs, you can elicit light –Applications becoming widely used in clinical diagnostics as a radioimmunoassay (RIA) or enzyme linked immunosorbent assay (ELISA) substitute some applicability to protein:protein and protein:DNA binding

26. BioSci 203 lecture 21 page 26 © copyright Bruce Blumberg 2001. All rights reserved Current generation binding assays (contd) Eletrochemiluminescence (contd) –advantages rapid and sensitive compared with RIA or ELISA good quantitation –disadvantages requires instrument proprietary reagents completely in vitro assay

27. BioSci 203 lecture 21 page 27 © copyright Bruce Blumberg 2001. All rights reserved Current generation binding assays (contd) Biacore (surface plasmon resonance) –surface plasmon waves are excited at a metal/liquid interface –Target bound to a thin metal foil and test sample flowed across it –Foil is blasted by a laser from behind SPR alters reflected light intensity at a specific angle and wavelength Binding to target alters refractive index which is detected as change in SPR Change is proportional to change in mass and independent of composition of binding agent

28. BioSci 203 lecture 21 page 28 © copyright Bruce Blumberg 2001. All rights reserved Current generation binding assays (contd) Biacore (contd) –Advantages Can use any target Biological extracts possible Measure kinetics Small changes detectable with correct instrument –360 d ligand binding to 150 kd antibody Can use as purification and identification system –Disadvantages Machine is expensive (we have two) “high throughput” very expensive Not trivial to optimize