OUTLINE Expression and manipulation of transgenes in the laboratory

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OUTLINEExpression and manipulation of
transgenes in the laboratory

• To study the protein Express your transgene
• Usually in E. coli, for speed, economy
• Expression in eukaryotic hosts
• Drive it with a promoter/enhancer
• Purify it via a protein tag
• Cleave it to get the pure protein
• In vitro mutagenesis to isolate variants of your
  protein or gene with desirable properties
• Single base mutations
• Deletions
• Cassette mutagenesis
• Explore protein-protein interaction
• Co-immunoprecipitation (co-IP) from
  extracts
• 2-hybrid formation
• surface plasmon resonance
• FRET (Fluorescence resonance energy
  transfer)
• Complementation readout

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Promoters for E. coli

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Inducible expression in E. coli T7 system
Component -IPTG IPTG
Lac repressor gene (I-gene) constitutively
on T7 RNA Polymerase gene driven by the lac
promoter off on Your favorite gene driven
by the T7 promoter off ON
Two-stage amplification of induction Many T7 Pol
molecules from one T7 pol gene Many YFG molecules
for every T7pol molecule Does not need to compete
with E. coli RNA pol all the T7 pol can be used
for the transgene transcription.
plasmid
T7 prom.
YFG
chromosome
IPTG
T7 RNA polymerase
Lac promoter
IPTG
X
Constitutive synthesis
chromosome
T7 RNA polymerase
Lac promoter
Lac I (repressor)
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His tag allows easy purification of the cloned
gene product
(Nitriloacetic acid chelate, NTA)
(competes with his)
Other cell proteins
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Assorted protein tags used for purification
Could be harmful
(poly-glucose)
(tripeptide incl cys)
Protein A is a bacterial protein that binds
tightly and specifically to IgG Avidin from egg
white binds biotin strongly (Kd10-15).
Streptavidin similar, from bacteria, cleaner
(less charged, not glycosylated) Proteins can be
biotinylated in vivo by bacterial enzymes acting
on specific peptide substrates (at lys) MBP
maltose binding protein GST glutathione-S-transf
erase
E-Xaa-Xaa-Y -Xaa-Q-(G/S),
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Cleave a fusion protein (like GST-fusions) using
TEV (tobacco etch virus) protease Insert a
specific substrate sequence between the 2 protein
domains (E-Xaa-Xaa-Y -Xaa-Q-(G/S) highly
specific) Add enzyme. Separate pure protein.
Tagging protein GFP, GST, MBP, etc.
Protein of interest

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Tag removal after purification
OR
Enterokinase Asp Asp Asp Asp Lys
OR
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E. coli as a host

• Easy, flexible, high tech, fast, cheap but
  problems
• Folding (can misfold)
• Sorting -gt can form inclusion bodies
• Purification -- endotoxins
• Modification -- not done (glycosylation,
  phosphorylation, etc. )
• Glycoproteins
• Acylation acetylation, myristoylation
• Methylation (arg, lys)
• Phosphorylation (ser, thr, tyr)
• Sulfation (tyr)
• Prenylation (farnesyl, geranylgeranyl on cys)
• Vitamin C-Dependent Modifications (hydroxylation
  of proline and lysine)
• Vitamin K-Dependent Modifications (gamma
  carboxylation of glu)
• Selenoproteins (seleno-cys from ser tRNA at 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)
  (not discussed)
• In vitro (cell-free), for analysis only(good for
  radiolabeled proteins)

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Yeast Expression Vector
2mm-seq yeast ori oriE bacterial ori Ampr
bacterial selection LEU2 Leu biosynthesis
yeast selection
2mm plasmid
Gene ofinterest(GOI)
Complementation of an auxotrophy can be used
instead of drug-resistance
Saccharomyces cerevisiae(bakers yeast)
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Yeast -Genomic integration
HIS4
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)
Alcohol oxidase gene
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Half-genes
Linearized viral genome
Gene of interest
Strong polyhedron promoter
(Fall army worm)
Baculovirus as a vector
in insect cells
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Linearized viral genome
Half-gene
Constitutive early to late promoter
Half-gene
Strong polyhedron promoter
Gene of interest
Re-built ORF1629, required for viral growth
Re-built functional lacZ (blue)
Recombination to yield active virus
Gene of interest
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Baculovirus cont.
Incubate 2-3 days
Enterokinase
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In vitro translation following in vitro
transcription
Transcription to RNA via the T7 promoter T7 pol
Add ATP, GTP, tRNAs, amino acids, label
(35S-met), May need to add RNase
(Ca-dependent) to remove endogenous mRNA
VECTOR
cDNA
T7 RNA polymerase binding site (17-21 nt)
ACCATGG..
Labeled protein
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Mammalian cell expression Popular hosts cell
lines HeLa (human cevial cancer) HEK293 (human
embryonic kidney Vero (monkey) Cos
(monkey) Mouse 3T3 CHO (Chinese hamster ovary)
BHK (baby hamster kidney)
Generalized gene structure for mammalian
expression
polyA site
intron
cDNA gene
Mam. prom.
5UTR
3UTR
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Expression in mammalian cells HEK293 Human
embyonic kidney HeLa Human cervical
carcinoma\ CHO Chinese hamster
ovary Cos Monkey cells with makingSV40
replication proteins BHK Baby hamster
kidey HepG2 Human hepatoma 3T3 Mouse or human
exhibiting regulated (normal-like)
growth GH3 Rat pituitary cells PC12 Mouse
neuronal-like tumor cells MCF7 Human breast
cancer various others, many differentiated to
different degrees AND Primary cells cultured
with a limited lifetime. E.g., MEF mouse
embryonic fibroblasts HDF Human diploid
fibroblasts
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Expression in mammalian cells HEK293, HeLa, CHO,
cos, 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)
• Inducible / repressible tet, ecdysone,
  glucocorticoid (tet tetracycline)

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Got this far
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Regulated expression Tetracycline-reponsive
promoters Tet-OFF (add tet ? shut off)
VP16 tcnactn domain
tetRdomain
tTA tet activator fusion protein
active
No tetBinds tet operator(if tet not bound)
tetRdomain
VP16 tcnactn domain
Tetracycline (tet), or,better, doxicyclin (dox)
not active
polyA site
tTA cDNA
CMV prom.
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MINIMUM CMV prom.
Mutliple tet operator elements
tetRdomain
VP16 tcnactn domain
not active little transcripton (bkgd)
Tet (dox) present
MINIMUM CMV prom.
Remove tet (dox)
VP16 tcnactn domain
tetRdomain
active Plenty of transcripton (gt 50X)
RNA pol
MINIMUM CMV prom.
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Tetracycline-reponsive promoters Tet-ON (add tet
? turn on gene
tetRdomain
VP16 tcnactn domain
not active
Diff. fusion protein Does NOT bind tet
operator(if tet not bound)
tetRdomain
VP16 tcnactn domain
active
Tetracycline (tet), or,better, doxicyclin (dox)
polyA site
tTA cDNA
CMV prom.
Binds tet operator(if tet not bound)
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Tet-ON
MINIMUM CMV prom.
Mutliple tet operator elements
tetRdomain
VP16 tcnactn domain
not active (little transcription)
Tet (dox) absent
MINIMUM CMV prom.
Add tet (dox)
Tet or dox
active little transcripton (bkgd)
VP16 tcnactn domain
tetRdomain
active Plenty of transcripton (gt 50X)
RNA pol
MINIMUM CMV prom.
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In vitro site-specific mutagenesis
(of coli DNA polymerase)
M13 strand
M13 single stranded bacteriophage
50 WT
50 mutant
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Get rid of WT genome
Enzyme that removes U from U-containing DNA
Dut deoxyuracil phophotransferase. Dut- cells
accumulate dUTP, ? DNA Ung uracil
Nglycosylase. Ung- mutants do
not excise uracil from DNA Growth of plasmid
template plasmid on dut- ung- E. coli double
mutant cells ? U in DNA Synthesize mutant DNA
using T. Transfect heteroduplex into dutung E.
coli. U gets removed from WT template strand,
leaving apyrimidinic sites, these cannot
replicate. Mutant synthetic strand survives
normally.
Mutant E. coli allows U to persist in DNA
DpnI sites
Apyrimidinic DNA formed and will not replicate
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Use of DpnI to get rid of WT template.
Template WT methylated DpnI sites
DpnI
Synthesis
Newly synthesized mutant strand its DpnI sites
are not methyated,.
DpnI treatment selectively destroys the
hemimethylated WT strand
Mutant strand greatly enriched.
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PCR-ligation-PCR mutagenesis
Site to mutate
Note you can easily create an internal deletion
this way.
Ali SA, Steinkasserer A., Biotechniques. 1995
May18(5)746-50.
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RS1
RS2
Mutagenesis by overlap extension PCR
PCR ? fragment ? subsequent cloning in a plasmid
RS1
RS2
Cut with RE 1 and 2
Ligate into similarly cut vector
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Random mutagenesis but in a limited
region Cassette mutagenesis by error-prone PCR
Original sequence coding for, e.g., an enhancer
--------------------------------------------------
-------------------------------------------------
-------------------
PCR fragment with high Taqpolymerase and Mn
instead of Mg ? errors
-------------------------------------------
--------------------------------------------
-----------------
Cut in primer sites and clone upstream of a
reporter.
Pick colonies Analyze phenotypes Sequence
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Cassette mutagenesis by doped synthesis Target
e.g., an enhancer element
--------------------------------------------------
-------------------------------------------------
-------------------
Original enhancer sequence
---------------------------------------------
-------- ----------------------------------
-----------------
Buy 2 doped oligos anneal
Clone upstream of a reporter.
Doping e.g., 85 G, 5 A, 5 C, 5
T at each position
Pick colonies Analyze phenotypes Sequence
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Measuring protein-protein interactions in
vitro Xone protein Y another protein
Binding between (almost) purified
proteins- His6-X HA-Y Bind to nickel ion
column, elute, Western with HA Ab GST-X HA-Y
Bind to glutathione ion column, elute, Western
with HA Ab His6-X 35S-Y (made in vitro)
Bind Ni column, elute, gel autoradiography.
(HA flu hemagglutinin)
BIAcore (or several other measurements)
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Surface plasmon resonance (SPR) The binding
events are monitored in real-time and it is not
necessary to label the interacting biomolecules.
glass plate
http//home.hccnet.nl/ja.marquart/BasicSPR/BasicSp
r01.htm
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Surface plasmon resonance Detects binding of
protein or nucleic acids to molecules immobilized
on the opposite surface. Suitable for protein
protein interaction, measure kinetics, binding
constants. Use small amount (ug) and even ligands
in crude extracts Popular instrument Biacore
Flow cell carrying ligands or wash
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Protein-protein interactions in
cells Co-immunoprecipitation (co-IP) from
extracts 2-hybrid formation FRET
(Fluorescence resonance energy transfer) Complem
entation readout
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Co-immunoprecipitation Most times not true
precipitation, which requires about equivalent
concentrations. of antigen and antibody Use
protein A immobilized on beads (e.g., agarose
beads) Protein A from Staphylococcus aureus
binds tightly to Immunoglobulin G (IgG) from many
species.
Does X interact with Y in the cell?
incubate

anti-X IgG
Cell extract
Protein A

Wash by centrifugation Elute with SDS Detect X, Y
by Western blotting