Transcript Lab 6 Part B Powerpoint

PURIFICATION OF MFP
FROM AN OVERNIGHT CULTURE
LABORATORY 6
PART B
OVERVIEW:
• Goals:
• Explain confirmation of protein relates to
function of protein
• How does protein folding occur?
• Lab:
• Take cells growing in broth:
• Lyse (break open)
• From overnight LB/amp/ara culture
• Purify mFP from cell lysate
• using column chromatography
INTRODUCTION
• Once laboratories locate promising therapeutic
protein:
• Then locate and isolate gene that encodes the protein
• Insert gene into plasmid (to clone gene)
• Cloning vectors
• Plasmid engineered to replicate in high numbers
• Within bacterial cell
• Expression vectors
• pARA-R with rfp gene
• Plasmid engineered specifically for protein expression
• Transformed cells
• Allowed to express protein
• Lysed to release synthesized protein from cell
INTRODUCTION
• Mutant fluorescent protein:
• 238 aa in size
• Fluorophore located in center
• Highly hydrophobic
• In order to purify (separate) protein:
• Look for differences in hydrophobicity
• Hydrophobic verse hydrophilic
• Some have regions that are both
• Hydrophobic regions will “hide” in interior of molecule
• How to isolate a single protein?
• E. coli we are using produces HIGH concentrations of mFP
Separation uses protein folding
Unfolded
Folded−
INTRODUCTION
• Column chromatography
• Purification technique uses
hydrophobicity to separate and
purify proteins
• Plastic cylinder with resin
• Separating medium
• Contains small hydrophobic beads
• If mFP placed into solution of high
salt concentrations:
• mFP molecule distorted
• Hydrophobic regions adhere to resin
• Hydrophilic proteins then continue down
column and flushed away
INTRODUCTION
• mFP trapped in resin bed:
• Wash column with solution low salt
concentration
• Hydrophobic regions of mFP point
towards interior of molecule
• Will elute (wash out) moderately
hydrophobic molecules with buffer
• Use solution of very low salt
concentration to release mFP from
resin beads
Unfolded
Unfolded
Unfolded
Folded
Protein folding in binding buffer
SIDER: If a mutation
CONSc
IDE
hanges
R: If aan
mutation
amino
CONSIDE
ac
changes
id,
R: how
If a mutation
an
might
amino
this
cac
hanges
cid,
hange
how
an
affec
mig
am
in folding andprotein
proteinfolding
function?
and
protein
protein
folding
function?
and protein function?
!
fi c proteins areSeither
pecifi chydrophobic
proteins are
Spec
either
orifi
hydrophilic
c proteins
hydrophobic
,are either
or hydrophilic
hydrophobic
,
nding on the relative
depending
amount
on the
of depending
hydrophobic
relative amount
on
and
the
ofrelative
hydrophobic
amount
and
of hy
ophilic amino ac
hydrophilic
ids they contain.
aminohydrophilic
Hydrophobic
acids they camino
ontain.ac
Hydrophobic
ids they contain. Hy
eins and hydrophilic
proteins
proteins
and hydrophilic
can proteins
be separated
proteins
and hydrophilic
bycan be separated
proteinsby
can be
mn chromatography
column
. Incthis
hromatography
method,
column
a column
. In
chromatography
thisis
method, a.cIn
olumn
this method
is
Column
ed with small beads
packed
that
with
aresmall
coated
pac
beads
with
kedthat
with
a material
are
small
coated
beads
with
that
a material
are coated
Resin-coat
sin) that attracts
(ahydrophobic
resin) that attrac
amino
(a
tsres
hydrophobic
ac
in)
idsthat
, andattrac
the
amino
ts hydrophobic
acids, and the
amino
beads
ure of proteinsmixture
is dissolved
of proteins
and pas
mixture
s
is
ed
dis
over
solved
ofthe
proteins
and pas
issdis
ed s
over
olved
the
and pass
s. In order for the
beads
hydrophobic
. In order for
amino
beads
the hydrophobic
ac
. In
ids
order
to stic
for
k amino
the hydrophobic
acids to sticami
k
e resin, the proteins
to themus
resin,
t be
the
unfolded
proteins
to theto
res
mus
expos
in,
t the
be
eunfolded
the
proteins mus
to expos
t be unfold
e the
ophobic aminohydrophobic
acids, which tend
amino
hydrophobic
toac
be
ids
found
, whicon
h
amino
tend
the to
acids
be,found
which on
tend
the
to
e of the protein.
ins
C
ide
ertain
of the
saltprotein.
solutions
inside
Certain
called
of the
s
buffers
alt
protein.
solutions
Certain
called
salt
buffers
solutio
nfold proteinswill
to varying
unfold degrees
proteins
will
. tounfold
varying
proteins
degrees
to
. varying degrees.
ER 6
• In binding buffer,
hydrophobic proteins
unfold
!
!
• Unfolded hydrophobic
proteins adhere to the
hydrophobic column resin
• Folded hydrophilic
| S T UE
D–
E6
N T G U ICDHE A P T E R 6 | S T U D E N T G U I D E
proteins never adhere to
the column
es
Amgen
erved.Foundation. All
© 2013
rights
Amgen
reserved.
Foundation. All rights reserved.
Hydrophilic
proteins
!
Unfolded
Unfolded
Unfolded
Folded
Protein folding in wash buffer
SIDER: If a mutation
CONSc
IDE
hanges
R: If aan
mutation
amino
CONSIDE
ac
changes
id,
R: how
If a mutation
an
might
amino
this
cac
hanges
cid,
hange
how
an
affec
mig
am
ein folding andprotein
proteinfolding
function?
and
protein
protein
folding
function?
and protein function?
!
fi c proteins areSeither
pecifi chydrophobic
proteins are
Spec
either
orifi
hydrophilic
c proteins
hydrophobic
,are either
or hydrophilic
hydrophobic
,
nding on the relative
depending
amount
on the
of depending
hydrophobic
relative amount
on
and
the
ofrelative
hydrophobic
amount
and
of hy
ophilic amino ac
hydrophilic
ids they contain.
aminohydrophilic
Hydrophobic
acids they camino
ontain.ac
Hydrophobic
ids they contain. Hy
eins and hydrophilic
proteins
proteins
and hydrophilic
can proteins
be separated
proteins
and hydrophilic
bycan be separated
proteinsby
can be
mn chromatography
column
. Incthis
hromatography
method,
column
a column
. In
chromatography
thisis
method, a.cIn
olumn
this method
is
Column
ed with small beads
packed
that
with
aresmall
coated
pac
beads
with
kedthat
with
a material
are
small
coated
beads
with
that
a material
are coated
Resin-coat
sin) that attracts
(ahydrophobic
resin) that attrac
amino
(a
tsres
hydrophobic
ac
in)
idsthat
, andattrac
the
amino
ts hydrophobic
acids, and the
amino
beads
ure of proteinsmixture
is dissolved
of proteins
and pas
mixture
s
is
ed
dis
over
solved
ofthe
proteins
and pas
issdis
ed s
over
olved
the
and pass
s. In order for the
beads
hydrophobic
. In order for
amino
beads
the hydrophobic
ac
. In
ids
order
to stic
for
k amino
the hydrophobic
acids to sticami
k
e resin, the proteins
to themus
resin,
t be
the
unfolded
proteins
to theto
res
mus
expos
in,
t the
be
eunfolded
the
proteins mus
to expos
t be unfold
e the
ophobic aminohydrophobic
acids, which tend
amino
hydrophobic
toac
be
ids
found
, whicon
h
amino
tend
the to
acids
be,found
which on
tend
the
to
e of the protein.
ins
C
ide
ertain
of the
saltprotein.
solutions
inside
Certain
called
of the
s
buffers
alt
protein.
solutions
Certain
called
salt
buffers
solutio
nfold proteinswill
to varying
unfold degrees
proteins
will
. tounfold
varying
proteins
degrees
to
. varying degrees.
ER 6
• In wash buffer, moderately
hydrophobic proteins fold
!
!
• Highly hydrophobic
proteins, including RFP,
stay unfolded
• Folded moderately
hydrophobic proteins are
| S T UE
D–
E6
N T released
G U ICDHE A P T E R 6 | Sfrom
T U D E N T G the
U I D E column
es
Amgen
erved.Foundation. All
© 2013
rights
Amgen
reserved.
Foundation. All rights reserved.
Moderately
hydrophobic
proteins
!
ed
Unfolded
Folded
Folded
Folded
Protein folding in elution buffer
c
E
hanges
id,
R: how
If a mutation
an
might
amino
this
cac
hanges
c
id,
hange
how
an
affec
might
amino
t this
acid,
change
how might
affect this change affect
folding
n function?
and protein function?
!
!
!
drophilic
proteins
hydrophobic
,are either
or hydrophilic
hydrophobic
,
or hydrophilic,
ng
obic
amount
on
and
the
ofrelative
hydrophobic
amount
and
of hydrophobic and
hobic
ey
iliccamino
ontain.ac
Hids
ydrophobic
they contain. Hydrophobic
roteins
ated
and hydrophilic
bycan be separated
proteinsby
can be separated by
c
n
umn
hromatography
thisis
method, a.cIn
olumn
this method,
is
a column is
Column
Column
hat
with
a material
are
small
coated
beads
with
that
a material
are coated with a material
Resin-coated
Resin-coated
ophobic
sthat
, andattrac
the
amino
ts hydrophobic
acids, and the
amino acids, and the
beads
beads
olved
er
ofthe
proteins
and pas
issdis
ed s
over
olved
the
and passed over the
drophobic
n
ds
order
to stic
for
k amino
the hydrophobic
acids to sticamino
k
acids to stick
mus
es
expos
in,
t the
be
eunfolded
the
proteins mus
to expos
t be unfolded
e the
to expose the
whic
und
obicon
h
amino
tend
the to
acids
be,found
which on
tend
the
to be found on the
ain
ed
the
s
buffers
alt
protein.
solutions
Certain
called
salt
buffers
solutions called buffers
old
ying
proteins
degrees
to
. varying degrees.
ved.
• In elution buffer, highly
hydrophobic proteins,
including RFP, fold
!
!
!
• Folded highly
hydrophobic proteins,
including RFP, are
released from the
column
• RFP can be collected
RFP
Column
Resin-coated
beads
WHAT WILL YOU NEED TO DO?
• Preparation day 1 – lysing the cells
• Preparation day 2 – mFP purification using column
chromatography
Reasons for lysis
• Soluble proteins made by cell,
including red fluorescent protein, are
dissolved in cell cytoplasm
• Only way to access soluble proteins is
to lyse (break open) cell
• After lysis, soluble proteins can be
easily separated from insoluble
structural proteins
Reasons for separation
• Although the bacteria make a lot of red
fluorescent protein, there are up to 1,000
other proteins in a living cell
• Those other proteins might interfere with
intended use of RFP or of any other
protein you are isolating
• Pharmaceutical companies require
purified protein
WHAT WILL YOU NEED TO DO?
• Chromatography columns
•
•
•
•
Capped tightly
Stopcocks closed
Store upright to allow resin bed to form flat surface
Use ethanol to rinse resin if splashed on sides
• Open stopcock and let ethanol drain from column
• Leave about 2mm layer above resin bed
WHAT WILL YOU NEED TO DO?
• Chromatography columns
• Columns will have equilibration buffer
• (add 3000 µl equil. Buffer)
• Dispense down to 1 cm above resin
• Set up on ring stand for model
• High enough for collection below
• Make sure resin bed visible
• When done:
• Flush columns with 4-5 mL elution buffer
• Flush columns with 3 mL 20% ethanol
• Cap tightly!!!!
METHODS
• Do not let the supernatant (red) run
through the tube….once it is gone, it
is gone!!
• as soon as the red hits the first level
of the chromatography column tip,
get the 1.5 mL tube ready…collect
when the red drips!!
Purification of RFP from an overnight culture
Bruce Wallace
Overnight Cell pellet
culture
with RFP
Lysed
cells
Pellet
RFP with
cell debris binding buffer
Results
CONCLUSIONS
• What product do you have?
• Would this normally be the end?
• What's next??
• Now that have purified protein, run steps to be sure it is
purified
• Quality control, SDS Page, ELISA, Western Blot
• “fill and finish”
• Make into final form ready for distribution
• Ex: Enbr
• Two 20000 L tanks with bacteria
• Two 2L purified protein
• $2 million a Liter!!
IN SUMMARY:
• Rfp genemfp protein (highly hydrophobic)
• BB (Binding Buffer)- Unfolds hydrophobic mfp causing it to
adhere to the resin. Washes out all other very hydrophilic
elements
• WB (Wash Buffer)- Washes out the binding buffer and folded
moderately hydrophobic proteins.
• EB (Elution Buffer)- Folds highly hydrophobic proteins (mfp)
and releases them from the column last before washing out
any other remaining elements.
• Leaves you with just the purified mfp: red flourescent protein,
which glowed under UV light : )