1 1 E. coli as a host PROs: Easy, flexible, high tech, fast, cheap;...... but problems CONs Folding...
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Transcript of 1 1 E. coli as a host PROs: Easy, flexible, high tech, fast, cheap;...... but problems CONs Folding...
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E. coli as a host
• PROs: Easy, flexible, high tech, fast, cheap; . . . . . . but problems
• CONs
• Folding (can misfold)
• Sorting -> can form inclusion bodies
• Purification -- endotoxins• Modification -- not done (glycosylation, phosphorylation, etc. )
• Modifications:• Glycoproteins • Acylation: acetylation, myristoylation• Methylation (arg, lys)• Phosphorylation (ser, thr, tyr)• Sulfation (tyr)• Lipid addition (prenylation: farnesyl, geranylgeranyl, palmitoylation on cys;
myristoylation on N-terminus)• Vitamin C-dependent Modifications (hydroxylation of proline and lysine)• Vitamin K-dLipid additionnependent Modifications (gamma carboxylation of glu)• Selenoproteins (seleno-cys tRNA at UGA stop)
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Some alternative hosts
•Yeasts (Saccharomyces , Pichia)
•Insect cells with baculovirus vectors
•Mammalian cells in culture (later)
•Whole organisms (mice, goats, corn)
•In vitro (cell-free), for analysis only(good for radiolabeled proteins
33Yeast Expression Vector (example)
2 micron plasmid
2 micron seq:yeast orioriE = bacterial oriAmpr = bacterial selectionLEU2, e.g. = Leu biosynthesisfor yeast selection
Saccharomyces cerevisiae(baker’s yeast)
oriE
Your favorite
gene(Yfg)
LEU2
Ampr
GAPDterm
GAPDprom
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
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Genomic DNA
HIS4 mutation-
Yeast - genomic integration via homologous recombination
HIS4
gfY
pt Vector DNA
FunctionalHIS4 gene
DefectiveHIS4 gene
Yfg
tp
Genomic DNA
Homologous recombination
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Double recombination Yeast (integration in Pichia pastoris)
AOX1 gene (~ 30% of total protein)
Genomic DNA
AOX1p
Yfg
AOX1t HIS4 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 geneHis- host
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Protein-protein interactions
Yeast 2-hybrid systemYeast 3-hybrid and 1 hybrid systemsCo-immunoprecipitationPull-downsFar western blotsBiacore (surface plasmon resonance, SPR)Fragment complementation
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BD =(DNA) binding domain AD =activation domain UAS =upstream activating sequence
Yeast 2-hybrid system:To discover proteins that interact with each other, orTo test for interaction based on a hypothesis for a specific protein.
?
http://www.mblab.gla.ac.uk/~maria/Y2H/Y2H.html
(bait)
(prey)
Y = e.g., a candidate protein being tested for possible interaction with X
Or: Y = e.g., a cDNA library used to discover a protein that interacts with X
?
Positive control:
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Y = e.g., a cDNA library used to discover a protein that interacts with X
Recover the Y sequence from reporter+ colonies by PCR to idenify protein Y
No interaction between X and Y: no reporter expression
Yes, interaction between X and Y: reporter protein is expressed
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Fusion library
Two different assays help, as there are often many false positives.
http://www.mblab.gla.ac.uk/~maria/Y2H/Y2H.html
=“prey”
Bait protein is the known target proteinfor whom partners are sought
BD= DNA binding domain; TA = transactiavting domain
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3-HYBRID: select for proteins domains that bind a particular RNA sequence
Bait
Prey
Prey could be proteins from a cDNA library
Use a known tight protein-RNA interaction (e.g., from RNA phage MS2)
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Yeast one-hybrid:
Insert a DNA sequence upstream of the selectable or reporter
Transform with candidate DNA-binding proteins (e.g., cDNA library)fused to an activator domain.
Each T = one copy of a DNA target sequence
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Directed Evolution of a Glycosynthase via Chemical ComplementationHening Lin,† Haiyan Tao, and Virginia W. Cornish J. AM. CHEM. SOC. 2004, 126, 15051-15059
Turning a glycosidase into a glyco-synthase
Glycosidase: Glucose-Glucose (e.g., maltose) + H2O 2 Glucose
Indirect selection using a yeast 3-hybrid system:toward a more efficient glycosynthase enzyme
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Indirect selection using a yeast 3-hybrid system(one of the hybrid molecules here is a small molecule)
e.g., from a mutated library of enzyme glycosynthase genes
glucose
DHFR = dihydrofolate reductase GR = glucocorticoid receptor (trancription factor )MTX = methotrexate (enzyme inhibitor of DHFR)DEX = dexamethasone, a glucocorticoid agonist, binds to GRAD = activation domain, DBD = DNA binding domain
Leu2 geneLeu2 gene
Transform a yeast leucine auxotroph. Provide synthetic chimeric substrate molecules. Select in leucine-free medium.
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URA-3 (toxic)
Library of cellulase mutant genes(one per cell)
x x x x
cellulase
Survivors are enriched for cellulase genes that will cleave cellulose with greater efficiency (kcat / Km)
Yeast cell
Directed Evolution of Cellulases via Chemical Complementation. P. Peralta-Yahya, B. T. Carter, H. Lin, H. Tao. V.W. Cornish.
Selection of improved cellulases via the yeast 2-hybrid system
Cellobiose(disaccharide)
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Detail
16Pathway to pyrimidine nucleotides:
URA-3 = gene for orotidine phosphate (OMP) decarboxylase
5-fluoroorotic acid
5-fluoro-OMP
5-fluoro-UMP
RNA
URA-3 decarboxylation (pyr-4)
thymidylate synthetaseinhibitiondeath
How does the URA-3 “suicide” system work?
exogenousuridine
uridine kinase
analog
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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 ion column via X, elute (his), Western with HA Ab for Y
GST-X + HA-Y; Bind to glutathione ion column, elute (glutathione), Western with HA Ab
His6-X + 35S-Y (made in vitro); Bind Ni column, elute (his), gel + autoradiography. No antibody needed.
(HA = flu hemagglutinin) glutathione = gamma-glutamyl-cysteinyl-glycine.
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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)
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Western blotting
To detect the antibody use a secondary antibody against the primary antibody (e.g, goat anti-rabbit IgG).
The secondary antibody is a commercial fusion protein with an enzyme activity (e.g., alkaline phosphatase).
The enzyme activity is detected by its catalysis of a reaction producing a luminescent compound.
http://www.bio.davidson.edu/courses/genomics/method/Westernblot.html
*
*
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Y YNon-luminescent substrate-PO4
=
Luminescent product + PO4=
Protein band on membrane
Alkaline phosphatase fusion
Secondary antibody
Antibody to protein on membrane
Detect by exposing to film
Detection of antibody binding in western blots
(chemiluminescence)
21Far western blotting to detect specific protein-protein interactions. Use a specific purified protein as a probe instead of the primary antibody
To detect the protein probe use an antibody against it.
Then a secondary antibody against the first antibody, a fusion protein with an enzyme activity.
The enzyme activity is detected by its catalysis of a reaction producing a luminescent compound.
http://www.bio.davidson.edu/courses/genomics/method/Westernblot.html
protein protein
OR:Use a radioactively labeledprotein if interest and detect by autoradiography
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Expression via in vitro transcription followed by in vitro translation
cDNA
T7 RNA polymerasebinding site (17-21 nt)
….ACCATGG…..
VECTOR
2. Add a translation system: rabbit reticulocyte lysate or wheat germ lysate
Or: E. coli lysate (combined transcription + translation)
All commerically available as kits
Add ATP, GTP, tRNAs, amino acids, label (35S-met), May need to add RNase (Ca++-dependent) to remove endogenous mRNA In lysate
1. Transcription to mRNA via the T7 promoter + T7 polymerase
Radioactively labeled protein
NOTE: Protein is NOT at all pure (100s of lysate proteins present), just ~“radio-pure”
23Surface plasmon resonance (SPR)Popular instrument is a Biacore
The binding events are monitored in real-time and it is not necessary to label the interacting biomolecules.
http://home.hccnet.nl/ja.marquart/BasicSPR/BasicSpr01.htm
glass plate
Reflection angle changes depending on the mass of the material on the surface.Binding increases this mass. Follow as a functrion of concentration Kd’sOr time : Measure on-time, off time; Kd = off-time/on-time
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A Biacore result
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Got this far
26Expression in mammalian cellsLab examples: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 kidey HepG2 Human hepatomaGH3 Rat pituitary cellsPC12 Mouse neuronal-like tumor cellsMCF7 Human breast cancerHT1080 Human with near diploid karyotypeIPS induced pluripotent stem cells and:Primary cells cultured with a limited lifetime. E.g., MEF = mouse embryonic fibroblasts, HDF = Human diploid fibroblasts
Common in industry: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
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Mammalian cell expression
Generalized gene structure for mammalian expression:
cDNA geneMam.prom.
polyA site
intron
5’UTR3’UTR
Intron is optional but a good idea
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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)• Engineered inducible / repressible:
tet, ecdysone, glucocorticoid (tet = tetracycline)
29Engineered regulated expression:Tetracycline-reponsive promotersTet-OFF (add tet shut off)
tTA cDNA
tTA = tet activator fusion protein:tetRdomain
VP16 tc’nact’n domain
No tet.Binds tet operator(if tet not also bound)
tetRdomain
VP16 tc’nact’n domain
Tetracycline (tet), or,better, doxicyclin (dox)
active
not active
CMV prom.
polyA sitetTA gene must be in cell (permanent transfection, integrated):
Tet-OFF
Tet-OFF
(Bujold et al.)
Allosteric change in conformation
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MIN. CMV prom. your favorite gene
polyA site
Mutliple tet operator elements
MIN. CMV prom. your favorite gene
polyA site
tetRdomain
VP16 tc’nact’n domain
not activelittle transcripton (2%?, bkgd)
Doxicyclin present:
MIN. CMV prom. your favorite gene
polyA siteactivePlenty of transcripton
No doxicyclin:
tetRdomain
VP16 tc’nact’n domain
RNA po l
Tet-OFF
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Tetracycline-reponsive promotersTet-ON (add tet turn on gene
tTA cDNA
tetRdomain
VP16 tc’nact’n domain
tetRdomain
VP16 tc’nact’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
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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 siteactivePlenty of transcripton (> 50X)
Add dox:
tetRdomain
VP16 tc’nact’n domain
RNA pol II
Tet-ON
tetRdomain
VP16 tc’nact’n domain
not active little transcription (bkgd.)
doxicyclin
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SW Michnick web site: http://michnick.bcm.umontreal.ca/research/images/pca_general_en.gif
F = reporter protein fragment
Enzyme fragmentsthemselves do not associate well enough to reconstitute an active enzyme
Reporterenzyme
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FK506 = immunosuppressant drugFKBP = FK506 binding proteinFRAP = FKBP–rapamycin binding proteinFRB= FKBP–rapamycin binding domain of FRAP
DHFR = dihydrofolate reductaseDHF=dihydrofolate = FH2
THF=tetrahydrofolate = FH4
fMTX=fluorescent methotrexate
fMTX
DHFR fragments
Clonal selection and in vivo quantitation of protein interactions with protein-fragment complementation assays, I. Remy and S.W. Michnick PNAS 96, 394–5399, 1999
IN PURINE-FREE MEDIUM
Fluorescein-MTX
Rapamycin promotes the association of the 2 protein domains
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a phosphatase
FK506 recruits FKBP to bind to calcineurin and inhibit its action as a specific phosphatase
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Claim detection of 0.05 nM rapamycin??
No.
of
CH
O c
olon
ies
[rapamycin]
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Background association of FKBP and FRB without rapamycin
(compare)
Leucine zipper protein fragments instead ofrapamycin binding proteins (positive contro)
CHO cells(permanent transfection)
cos cells(transient transfection)
Fluorescent methotrexate (fMTX) assay
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8-fold increase in fluorescence per cell
Measure affinity for a drug in vivo
fluorescence-activated flow cytometer(FACS is this plus more)N
o. o
f ce
llsF
luor
esce
nce
inte
nsity
Log of fluorescence intensity
[rapamycin]
39
EMP1 = Erythropoietin mimetic peptide 1
Erythropoietin-erythropoietin receptor (dimer) interaction: Efficacy of a peptide mimetic
Erythropoietin
In vivo assay of drug effectiveness (EMP1)(inexpensive substitute for erythropoietin?)
Erytropoietin (EPO) receptor
40
FACS = Fluorescence-activated cell sorter
Impart a charge on the recognized cell
Less than one cell or particle per droplet. Thus the most that most droplets contain is one particle.
Charged plates attract droplets containing a particle of the opposite charge
Cells remain viable if treated with care.
Can be used purely anaytically without the sorting capability. Thencalled “flow cytometry”, or also called FACS anyway.
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No.
of
cells
Having this much fluorescence
Histogram-type display
No fluorescence (background autofluorescence)
Red stained
Usually a log scale
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One cell
Amount of red fluorescence (log)
Am
ount
of
gre
en f
luor
esce
nce
(log)
Say, want high reds butlow greens:Instruct the FACS to deflect cells in this quadrant only. Collect and grow or analyze further.
Analysis on 2 colors
Scatter plot display
You decide on the positions of of demarcations
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A. Flow cytometry data: 2-D plots where each point represents one particle. Then contour lines plotted around the point density. Here light “forward” scattering (irrespective of wavelength) is measured (FSC). Instrument can be set to reject data from 2-bead doublets that scatter light more.
B-D. Amplified beads hybridized to 2 probes, one specific to the S allele of a certain gene and one specific to the L allele. The beads carry the amplified PCR products corresponding to this region from 3 human individuals. The blue points come from microspheres that contained both types of PCR products from both alleles, despite the high dilution.
Green signalR
ed s
ign
al
Both signals
Neither signal
Analysis of beads representing the human genome using allele-specific hybridization probes and the FACS
Beaming bead FACS analysis
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