E. coli as a host

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E. coli as a host

• PROs Easy, flexible, high tech, fast, cheap .
  . . . . . but problems
• CONs
• Folding (can misfold)
• Sorting -gt 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

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Yeast Expression Vector (example)
Saccharomyces cerevisiae(bakers yeast)
2 micron seq yeast ori oriE bacterial ori Ampr
bacterial selection LEU2, e.g. Leu
biosynthesisfor yeast selection
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
GAPD the enzyme glyceraldehyde-3 phosphate
dehydrogenase
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Yeast - genomic integration via homologous
recombination
HIS4
Homologous recombination
t
p
Yfg
FunctionalHIS4 gene
DefectiveHIS4 gene
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Double recombination Yeast (integration in Pichia
pastoris)
P. pastoris-tight control-methanol induced
(AOX1)-large scale production (gram
quantities)
His- host
Alcohol oxidase gene
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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
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Yeast 2-hybrid system To discover proteins that
interact with each other, or To test for
interaction based on a hypothesis for a specific
protein.
Positive control
(bait)
?
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
?
(prey)
BD (DNA) binding domain AD activation domain
UAS upstream activating sequence
http//www.mblab.gla.ac.uk/maria/Y2H/Y2H.html
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No interaction between X and Y no reporter
expression
Yes, interaction between X and Y reporter
protein is expressed
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
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Fusion library
Bait protein is the known target proteinfor whom
partners are sought
prey
Two different assays help, as there are often
many false positives.
BD DNA binding domain TA transactiavting
domain
http//www.mblab.gla.ac.uk/maria/Y2H/Y2H.html
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3-HYBRID select for proteins domains that bind
a particular RNA sequence
Use a known tight protein-RNA interaction (e.g.,
from RNA phage MS2)
Prey
Bait
Prey could be proteins from a cDNA library
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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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Indirect selection using a yeast 3-hybrid
systemtoward a more efficient glycosynthase
enzyme
Directed Evolution of a Glycosynthase via
Chemical Complementation Hening Lin, Haiyan Tao,
and Virginia W. Cornish J. AM. CHEM. SOC. 2004,
126, 15051-15059
Turning a glycosidase into a glyco-synthase Glyco
sidase Glucose-Glucose (e.g., maltose) H2O
? 2 Glucose
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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
Leu2 gene
Leu2 gene
Transform a yeast leucine auxotroph. Provide
synthetic chimeric substrate molecules. Select
in leucine-free medium.
DHFR dihydrofolate reductase GR
glucocorticoid receptor (trancription factor
) MTX methotrexate (enzyme inhibitor of
DHFR) DEX dexamethasone, a glucocorticoid
agonist, binds to GR AD activation domain, DBD
DNA binding domain
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Selection of improved cellulases via the yeast
2-hybrid system
Survivors are enriched for cellulase genes that
will cleave cellulose with greater efficiency
(kcat / Km)
Yeast cell
Cellobiose (disaccharide)
URA-3 (toxic)
cellulase
Directed Evolution of Cellulases via Chemical
Complementation. P. Peralta-Yahya, B. T. Carter,
H. Lin, H. Tao. V.W. Cornish.
x
x
x
x
Library of cellulase mutant genes (one per cell)
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Detail
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Pathway to pyrimidine nucleotides
How does the URA-3 suicide system work?
analog
5-fluoroorotic acid

• URA-3 gene for orotidine phosphate (OMP)
  decarboxylase

5-fluoro-OMP
URA-3 decarboxylation (pyr-4)
5-fluoro-UMP
uridine kinase
exogenousuridine
thymidylate synthetase inhibition
RNA
death
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Measuring protein-protein interactions in vitro
Xone 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
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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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/metho
d/Westernblot.html
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Detection of antibody binding in western blots
Antibody to protein on membrane
Alkaline phosphatase fusion
Non-luminescent substrate-PO4
Y
Y
Luminescent product PO4
(chemiluminescence)
Secondary antibody
Detect by exposing to film
Protein band on membrane
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Far 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.
protein
protein
ORUse a radioactively labeledprotein if
interest and detect by autoradiography
http//www.bio.davidson.edu/courses/genomics/metho
d/Westernblot.html
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Expression via in vitro transcription followed by
in vitro translation
T7 RNA polymerase binding site (17-21 nt)
VECTOR
cDNA
.ACCATGG..
Radioactively labeled protein
1. Transcription to mRNA via the T7 promoter T7
polymerase
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
NOTE Protein is NOT at all pure (100s of lysate
proteins present), just radio-pure
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Surface 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.
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
? Kds Or time Measure on-time, off time Kd
off-time/on-time
http//home.hccnet.nl/ja.marquart/BasicSPR/BasicSp
r01.htm
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A Biacore result
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• Got this far

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Expression in mammalian cells Lab
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 (-gt 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 hepatoma GH3 Rat pituitary
cells PC12 Mouse neuronal-like tumor
cells MCF7 Human breast cancer HT1080 Human with
near diploid karyotype IPS 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 cells Vero vaccines
African greem monkey cells CHO Mabs, other
therapeutic proteins Chinese hamster ovary
cells PER6 Mabs, other therapeutic proteins Human
retinal cells
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Mammalian cell expression
Generalized gene structure for mammalian
expression
polyA site
intron
Mam.prom.
cDNA gene
3UTR
5UTR
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)

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Engineered regulated expression Tetracycline-repo
nsive promoters Tet-OFF (add tet ? shut off)
Tet-OFF
VP16 tcnactn domain
tetRdomain
tTA tet activator fusion protein
active
No tet.Binds tet operator(if tet not also bound)
Tet-OFF
VP16 tcnactn domain
tetRdomain
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.)
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Tet-OFF
MIN. CMV prom.
Mutliple tet operator elements
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Tet-ON
Tetracycline-reponsive promoters Tet-ON (add tet
? turn on gene
tetRdomain
VP16 tcnactn domain
not active
Different fusion protein Does NOT bind tet
operator(if tet not bound)
tetRdomain
VP16 tcnactn 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
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Tet-ON
MIN. CMV prom.
Mutliple tet operator elements
tetRdomain
VP16 tcnactn domain
not active little transcription (bkgd.)
Doxicyclin absent
MIN. CMV prom.
Add dox
active
tetRdomain
VP16 tcnactn domain
doxicyclin
active Plenty of transcripton (gt 50X)
RNA pol II
MIN. CMV prom.
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Reporterenzyme
Enzyme fragments themselves do not associate well
enough to reconstitute an active enzyme
F reporter protein fragment
SW Michnick web site http//michnick.bcm.umontrea
l.ca/research/images/pca_general_en.gif
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Clonal selection and in vivo quantitation of
protein interactions with protein-fragment
complementation assays, I. Remy and S.W. Michnick
PNAS 96, 3945399, 1999
DHFR fragments
Rapamycin promotes the association of the 2
protein domains
fMTX
Fluorescein-MTX
IN PURINE-FREE MEDIUM
DHFR dihydrofolate reductase DHFdihydrofolate
FH2 THFtetrahydrofolate FH4 fMTXfluorescent
methotrexate
FK506 immunosuppressant drug FKBP FK506
binding protein FRAP FKBPrapamycin binding
proteinFRB FKBPrapamycin binding domain of
FRAP
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FK506 recruits FKBP to bind to calcineurin and
inhibit its action as a specific phosphatase
a phosphatase
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Claim detection of 0.05 nM rapamycin ??
No. of CHO colonies
rapamycin
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Fluorescent methotrexate (fMTX) assay
CHO cells (permanent transfection)
cos cells (transient transfection)
Leucine zipper protein fragments instead
of rapamycin binding proteins (positive contro)
Background association of FKBP and FRB without
rapamycin
(compare)
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8-fold increase in fluorescence per cell
No. of cells
fluorescence-activated flow cytometer (FACS is
this plus more)
Log of fluorescence intensity
Fluorescence intensity
Measure affinity for a drug in vivo
rapamycin
Competition with a molecule that binds only one
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Erythropoietin-erythropoietin receptor (dimer)
interaction Efficacy of a peptide mimetic
Erytropoietin (EPO) receptor
In vivo assay of drug effectiveness
(EMP1) (inexpensive substitute for
erythropoietin?)
EMP1 Erythropoietin mimetic peptide 1
Erythropoietin
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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.
Can be used purely anaytically without the
sorting capability. Then called flow cytometry,
or also called FACS anyway.
Charged plates attract droplets containing a
particle of the opposite charge
Cells remain viable if treated with care.
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Histogram-type display
No fluorescence (background autofluorescence)
No. of cells
Red stained
Usually a log scale
Having this much fluorescence
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Scatter plot display
Analysis on 2 colors
One cell
Amount of green fluorescence (log)
You decide on the positions of of demarcations
Say, want high reds but low greens Instruct the
FACS to deflect cells in this quadrant only.
Collect and grow or analyze further.
Amount of red fluorescence (log)
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Beaming bead FACS analysis
Analysis of beads representing the human genome
using allele-specific hybridization probes and
the FACS
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.
Both signals
Red signal
Neither signal
Green signal
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.
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