C-LYTAG Purification System Biomedal

C-LYTAG
Purification System
Ed. 1
Web: www.biomedal.com
.
© 2006 Biomedal, S.L.
Email: info@biomedal.com
User's manual
Biomedal
C-LYTAG PURIFICATION SYSTEM
A system for immobilization and single step purification of recombinant
proteins.
For research use only
1. About C-LYTAG system .................................................2
2. Strain and vectors ......................................................4
2.1. E. coli REG-1 strain .........................................4
2.2. pALEX vectors ..................................................5
2.2.1. pALEX map ...................................................5
2.2.2. General information...................................5
2.2.3. Sequence and restriction analysis ..............6
3. Protocols ....................................................................8
3.1. Cloning into pALEX vectors and host strain
transformation (E. coli REG-1) ...........................8
3.2. Protein expression ............................................9
3.3. Protein purification ...........................................9
3.3.1. Column preparation ...............................10
3.3.2. Extract cell preparation ...........................10
3.3.3. Washing and elution .................................11
3.3.4. Batch purification ...................................11
3.3.5. Choline elimination ................................12
3.3.6. Enterokinase cleavage .............................12
3.3.7. Column regeneration and storage ...........12
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4. Appendix .................................................................14
4.1. Composition of buffers ....................................14
4.2. Characteristics of C-LYTRAP resin .....................15
4.3. Troubleshooting .............................................15
4.4. Related products ............................................16
4.5. References .....................................................17
C-LYTAG PURIFICATION SYSTEM
Contents
1
1. About C-LYTAG system
2
C-LYTAG system enables the single-step affinity
purification of C-LYTAG fusion proteins using C-LYTRAP
resin, a simple, selective and cost efficient chromatographic
support. Binding conditions are gentle and do not involve
covalent modifications, therefore the fusion protein is
Figure 1. C-LYTAG structure
highly stable once bound to the resin. For this reason,
the system can also be used for enzyme immobilization onto solid supports.
C-LYTAG-fusion proteins are selectively eluted using choline-containing buffers.
C-LYTAG system yields high expression levels when induced, but maintains
basal levels prior to induction. It integrates CASCADE™* technology, in which
the expression of the fusion protein is controlled by linked regulatory circuits
to amplify gene expression (Figure 2) (4). CASCADE™ employs two salicylateresponsive transcriptional activator proteins, NahR and XylS2. In the presence
of salicylate, the NahR protein induces expression of XylS2 from the Psal promoter.
Salicylate also activates XylS2, which induces high-level expression of the gene
of interest from the Pm promoter. The synergistic effect of using two transcriptional
regulators in a sequential cascade amplifies expression levels nearly 20-fold
compared to expression from either promoter individually.
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C-LYTAG PURIFICATION SYSTEM
C-LYTAG is a system comprising an integrated range
of products for the expression and purification of CLYTAG fusion proteins in E. coli. The method is based
on the selective interaction of the choline binding domain
of the Streptococcus pneumoniae N-acetylmuramoyl-Lalanine amidase LytA (C-LYTAG) with choline or choline
analogues (tertiary or quaternary amines)(1,2). The
macromolecular structure of C-LYTAG has been resolved
(Figure 1) (3).
NOTES
NOTES
The first portion of the cascade system (nahR/Psal::XylS22) is located in the
chromosome of the E. coli REG-1 expression strain and the second (Pm/lacO/CLytA) is contained in pALEX vectors.
Pm
xylS2
gene of interest
Salicylate
Figure 2. CascadeTM Expression system
C-LYTAG PURIFICATION SYSTEM
nahR
Psal
The C-LYTAG Purification System (Cat. No. KT-3246) components are:
Component
pALEXa
pALEXb
pALEXc
E. coli REG-1 strain
Inducer (salicylate, 1M)
C-LYTRAP
Choline chloride (3M)
Quantity
8 mg (each)
Stab
25 ml
30 ml
20 ml
Storage
-20ºC
CAT. Nº.
EV-3240
EV-3241
EV-3242
4ºC
BS-3262
4ºC
RS-3247
4ºC; EtOH 20% RS-3302
4ºC
RS-3245
NOTE: C-LYTAG Purification System is shipped at room temperature. Upon arrival, store the
components according to the directions in the table above.
* CASCADE™ is a trade mark of Active Motif, Inc., Carlsbad. The CASCADE™ expression
system is patent pending and licensed by Active Motif, Inc. Commercial license available. Please
contact us if you want more information about license agreement.
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3
20ml
RS-3245
Salicylate (1M)
Inductor.
25ml
RS-3247
Cell culture tested.
5g
RS-3217
Kanamycin
Cell culture tested.
5g
RS-3219
ANTIBODY
Anti C-LYTAG
Polyclonal antibody.
100 ml
AB-3238
OTHERS
Multibind 96
C-LYTAG
Multi-well format for multiple
assays and high throughput
screenings of biomolecules
tagged with C-LYTAG
1 plate
RS-3317
5 plates
RS-3318
C-LYTRAP Spin
columns
Microcentrifuge columns
containing C-LYTRAP resin
10 units
RS-3319
15 units
RS-3320
100 units
RS-3321
1 unit
(reusable)
RS-3322
1. One-step purification from crude lysate to >95% pure protein.
2. Tightly regulated expression.
3. Resin is simple, inexpensive and reusable.
4. Compatible with virtually all common buffers.
5. Purified fusion protein can rebind to the matrix.
6. Elution buffers do not interfere with protein quantitation (Coomassie,
UV-absorption, etc.).
ANTIBIOTICS Ampicillin
7. The C-LYTAG moiety is easily refolded from inclusion bodies into a
functional conformation.
8. No covalent modification of the protein is needed for efficient
immobilization.
9. It has been successfully tested in many cases, and in a wide range of
4
protein sizes: from small peptides (<10 aa) to large proteins (>1000
aa).
2. Strain and vectors
2.1. E. coli REG-1 strain
E. coli REG-1 strain contains the regulatory element nahR/Psal::xylS2 integrated
into the chromosome.
Genotype
Mini-Tn5(kanr nahR/Psal::xylS2) mcrA D(mrr-hsdRMS-mcrBC) M80lacZDM15
DlacX74 recA1 araD139 (ara-leu)7697 galU galK rpsL endA1 nupG.
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Purification
column
Empty columns for
C-LYTRAP packing
4.5. References
1.- Sanz et al (1988). FEBS Lett. 232, 308-312.
2.- Sánchez-Puelles et al (1992). Eur. J. Biochem. 203, 153-159.
3.- Fernández -Tornero et al (2002). J. Mol. Biol. 321, 163-173.
4.- Cebolla et al (2001). Nucleic Acids Res. 29, 759-766.
5.- CascadeTM. Instruction Manual. Active Motif.
C-LYTAG PURIFICATION SYSTEM
Choline chloride Preparation of elution buffer.
3M
Main advantages of C-LYTAG Purification System
17
2.2. pALEX vectors
Fusion protein binds to
C-LYTRAP but does not elute.
Reduce the flow rate or let the
elution buffer and resin in
contact for 30 min.
Non-specific interactions
with the support.
Add 1.5% Triton X-100 to the
elution buffer.
Ionic interactions of the
target protein with the
support.
Increase the ionic strength in
the choline-containing elution
buffer (up to 1.5 M).
Fusion protein precipitation.
Fusion protein forms dimers.
2.2.1. pALEX map
C-LYTAG PURIFICATION SYSTEM
Elution is too fast.
Add non-denaturing solubilizing
agents (mild detergents, etc.).
Choline absence.
Do not remove choline.
Choline is present, which
induces dimerization of
C-LYTAG.
Remove choline.
4.4. Related products
PRODUCT
STRAINS
E. coli REG-1
DESCRIPTION
Propagation of pALEX vectors
and protein expression.
FORMAT
Stab
Cat.No.
BS-3262
PRIMERS
C-LYTAG primer
Amplification by PCR
(selection of positive clones)
and sequencing of
pALEX vectors.
2 nmol
PR-3281
pALEXa
Cloning and expression
of C-LYTAG fusion proteins.
8 mg
EV-3240
2.2.2. General information
pALEXb
Cloning and expression of
C-LYTAG fusion proteins.
8 mg
EV-3241
pALEXc
Cloning and expression of
C-LYTAG fusion proteins.
8 mg
EV-3242
pALEX vectors are derivatives of pCAS vectors (5) and contain the 3´ moiety
of the Streptococcus pneumoniae lytA gene (C-LYTAG protein) between Pm
promoter/lac operator and the multiple cloning site.
pALEX-lacZ
Positive control.
8 mg
EV-3243
pALEX-gfplav
Positive control.
8 mg
EV-3239
pALEX-lip36
Positive control.
8 mg
EV-3244
TSS
Preparation of competent cells.
1.5 ml
RS-3215
1.5 mlx5
RS-3216
30 ml
RS-3302
250 ml
RS-3316
16
VECTORS
REAGENTS
AND
SOLUTION
C-LYTRAP
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Resin for C-LYTAG protein
purification.
5
pALEX vectors contain a multiple cloning site (MCS) that has seven unique
restriction sites: BamHI, XhoI, SacI, BglII, KpnI, BstBI and HindIII (refer to vector
map for details). They are available in all three reading frames (pALEXa, pALEXb
and pALEXc), to facilitate cloning.
pALEX vectors also carry an enterokinase recognition sequence that enables
removal of the C-LYTAG moiety from the fusion protein.
pALEX vectors carry as selection marker the bla gene, that confers ampicillin
resistance.
2.2.3. Sequence and restriction analysis.
4.2. Characteristics of C-LYTRAP resin
pALEXa vector
XbaI
Binding capacity
Bead structure
Bead size
Recommended flow rate
pH stability (<2 h)
pH stability (> 2 h)
Antimicrobial agent
Storage
0.5-3 mg/ml
6% highly cross-linked agarose
45-165 mm
1 ml/min
1-14
3-12
Ethanol 20%
4ºC for long time periods
201 TGCAAGAAGC GGATACAGGA GTGCAAAAAA TGGCTATCTC TAGAAAGGCC
251 TACCCCTTAG GCTTTATGCA ACAGAAACAA TAATAATGGA GTCATGACCA
4.3. Troubleshooting
301 TGACAATGCA CCTGGGGCTC GACTATATAG ATAGTCTCGT TGAAGAAGAT
351 GAGAACGAGG GCATCTACCG CTGCAAGCGC GAGATGTTCA CCGACCCTCG
401 GCTGTTCGAT TTAGAGATGA AACACATCTT TGAGGGCAAC TGGATTTATC
PROBLEM
POSSIBLE CAUSE
RECOMMENDATION
No expression or low
expression levels.
Gene is cloned into the
wrong reading frame.
Review your cloning strategy to
ensure that you choose the
correct a, b or c vector.
Culture temperature is too
high or too low.
Change culture temperature.
Concentration of inducer
is too low.
Increase the concentration of
inducer (0.5-5 mM salicylate).
451 TCGCCCACGA GAGCCAGATT CCCGAGAAGA ACGACTATTA CACCACGCAG
6
501 ATGGGCCGGC AGCCGATATT CATCACACGC AACAAAGATG GTGAGCTGAA
EcoRI
Messenger RNA instability
Overexpression of the
or problems with translation corresponding tRNA can help
(presence of multiple rare
codons in the gene of
interest).
551 TGCCTTCGTC AATGCCTGAA TTCGGAATTG TGAGCGGATA ACAATTCCTA
601 ACTTTATAGA TTACAAAACT TAGGAGGGTT TTTACCATGA TGGGCATTAG
M M
RBS
G I S
651 CCGTGAGCAG TTTAAGCATG ATATTGAGAA CGGCTTGACG ATTGAAACAG
R E
Q
F
K
H
D
I
E
N
G
L
T
I
E
T
G
701 GCTGGCAGAA GAATGACACT GGCTACTGGT ACGTACATTC AGACGGCTCT
W
Q
N
K
D
T
G
Y
W
Y
V
H
S
D
G S
751 TATCCAAAAG ACAAGTTTGA GAAAATCAAT GGCACTTGGT ACTACTTTGA
Y
P
K
D
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K
F
E
K
I
N
G
T W Y
Y
F D
Insufficient cell breaking.
Check cell breaking conditions.
Improper protein folding.
Culture temperature is too
high.
Use a lower temperature (30ºC
or less).
Fusion protein is not retained
by the resin.
The amount of resin is not
adequate.
Add more resin.
Tertiary or quaternary
amines in the extract buffer.
Dilute the extract so that amine
concentration is below 10 mM.
Steric hindrance between
C-LytA and the fused
protein.
Add a short peptide between
both peptidic fragments.
C-LYTAG PURIFICATION SYSTEM
It is indicated:
ORANGE: Pm Promoter
PALE BLUE: Variable region in pALEXa, b and c vectors
RED: C-LYTAG sequence
BLUE: Enterokinase recognition sequence
: pALEX Primer Forward (Cat. NO. PR-3433)
: pALEX Primer Reverse (Cat. NO. PR-3434)
15
4. Appendix
801 CAGTTCAGGC TATATGCTTG CAGACCGCTG GAGGAAGCAC ACAGACGGCA
4.1. Composition of buffers
Cell resuspension buffer
S
20 mM sodium phosphate pH 7.0
BUFFERS FOR INCLUSION BODIES TREATMENT
guanidine chloride 6 M solution
Buffer 2:
20 mM sodium phosphate pH 7.0
150 mM choline chloride
1.5 M guanidine chloride
Buffer 3:
Buffer 4:
20 mM sodium phosphate pH 7.0
150 mM choline chloride
0.5 M guanidine hydrochloride
20 mM sodium phosphate pH 7.0
Y
M
L
A
D
R
W
R
K
H
T
D
G
N
851 ACTGGTACTG GTTCGACAAC TCAGGCGAAA TGGCTACAGG CTGGAAGAAA
W
Y
F
W
D
N
S
G
E
M
A
T
G
W
K
K
901 ATCGCTGATA AGTGGTACTA TTTCAACGAA GAAGGTGCCA TGAAGACAGG
I
A
D
K
W
Y
Y
F
N
E
E
G
A
M
K
T
G
951 CTGGGTCAAG TACAAGGACA CTTGGTACTA CTTAGACGCT AAAGAAGGCG
W
V
K
Y
K
D
T
W
Y
Y
L
D
A
K
E
G
A
NcoI
1001 CCATGGTATC AAATGCCTTT ATCCAGTCAG CGGACGGAAC AGGCTGGTAC
M
V
S
N
A
F
I
Q
S
A
D
G
T
G
W
Y
EcoRI
1051 TACCTCAAAC CAGACGGAAC ACTGGCAGAC AGGCCAGAAT TCACAGTAGA
Y
L
K
P
D
G
T
L
A
D
R
P
E
F
T
V
E
NheI
PURIFICATION BUFFERS
14
G
Column equilibration buffer:
20 mM sodium phosphate pH 7.0
Washing buffer:
20 mM sodium phosphate pH 7.0
1.5 M NaCl
Re-equilibration buffer:
20 mM sodium phosphate pH 7.0
150 mM NaCl
Elution buffer:
20 mM sodium phosphate pH 7.0
150 mM choline chloride
NOTE :
1101 GCCAGATGGC TTGATTACAG TAAAAGCTAG CATGACTGGT GGACAGCAAA
P
D
G
L
I
T
V
K
A
S
M
T
G
G
EK Cleavage Sites ClaI
Q
Q M
BamHI SacI
1151 TGGGTCGGGA TCTGTACGAC GATGACGATA AGGATCGATG GGGATCCGAG
G
R
D
L
BglII
XhoI
Y
D
D
D
D
K
D
PvuII
R
W
G
BstBI
PstI
KpnI
NdeI
EcoRI
S
E
NotI
HindIII
1201 CTCGAGATCT GCAGCTGGTA CCATATGGGA ATTCGAAGCT TGCGGCCGCC
L
E
I
C
S
W
Y
H
M
G
I
R
S
L
R
P
P
a. Salt (up to 1.5M NaCl) may be added to the cell resuspension and column equilibration
buffers. The presence of the salt excludes non C-LYTAG containing proteins and
nucleic acids from binding to the resin. When determining optimal salt concentrations,
the stability of the recombinant protein must be considered in relation to the ionic
strength of the buffers.
b. Streptomycin sulfate (40 mg/ml) may be added to the resuspension buffer. Its addition
causes the precipitation of nucleic acids, reduces the viscosity of the extract and
decreases non-especific binding to the column.
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C-LYTAG PURIFICATION SYSTEM
Buffer1:
S
1251 CAGCTTGCTG GCGTACCGTT CCTGTCTAAA ATCCCTTTAA TCGGCCTCCT
S
L
L
A
Y
R
S
C
L
K
S
L
*
Version B variable region: GATCCGAG
Version C variable region: CATCGATG GATCCGAC
Note: Restriction sites ClaI, PstI, EcoRI and NotI are not uniques in the plasmid. You
should take into account this in order to design your cloning strategy
7
RESTRICTION ENZYMES THAT DO NOT CUT pALEX vectors
AscI
AsiSI
AvrII
BbvCI
BclI
BfrBI
BlpI
BmgBI
BplI
Bpu10I
BseRI
BsgI
BsiWI
BsmBI
BspEI
BspMI
BsrGI
BssHI
BstEII
BstZ17I
EcoICRI*
EcoNI
EcoRV
FalI
FseI
FspAI
HpaI
MfeI
MluI
MscI
NruI
NsiI
PacI
PflMI
PfoI
PmeI
PmlI
PshAI
Psp0MI
RsrII
SacI*
SacII
SalI
SapI
SbfI
SexAI
SfiI
SgrAI
SmaI
SpeI
SphI
SrfI
SwaI
Tth111I
XcmI
XmaI
* pALEXc only
GENERAL NOTES:
RESTRICTION ENZYMES THAT CUT ONCE pALEX vectors
AatII
Acc65I
AflIII
AhdI
AloI
AlwNI
BaeI
BamHI
BbeI
BglI
BglII
BmtI
BsaXI
BsmI
BsmFI
BstAPI
BstBI
Bsu36I
BtgI
Drolll
Eorl
EcolCRI
Fspl
HincII
HinDIIl
Kasl
Kpnl
NarI
NcoI
NdeI
NheI
NspI
PciI
PpuMI
PsiI
PsrI
PvuI
PvuIl
SacI
SanDI
ScaI
SfoI
SnaBI
StuI
StyI
XbaI
XhoI
ZraI
a. The purification can be performed at 4ºC or room temperature, depending
on the stability of the target protein.
b. The re-equilibration procedure reduces the presence of salts in the purified
fraction and may eliminate the presence of nucleic acids or other
components of the crude extracts which could otherwise be eluted by
the small, but significant, additional increase in the ionic strength
produced when choline is added.
c. The presence of choline analogues (tertiary and quaternary amines) in
a relatively high concentration (>20 mM) may prematurely elute the
8
target protein.
3. Protocols
3.1. Cloning into pALEX vectors and host strain transformation
(E. coli REG-1)
The cloning strategy must take into consideration that:
- The DNA encoding the target protein must be cloned in frame with the start
codon (ATG) of the C-LYTAG coding sequence (see C-LYTAG and MCS
sequence). Determine which restriction sites will be used for cloning and then
choose the pALEX vector that will preserve the reading frame at the 5´ end.
- A stop codon must be included to terminate protein translation. pALEX vectors
do not include a stop codon.
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C-LYTAG PURIFICATION SYSTEM
AarI
AccI
AfeI
AflII
AgeI
AleI
ApaI
If contaminants are present:
1. Wash with 5 volumes NaOH 1 M.
2. Wash with 5 volumes of water.
3. Wash with 5 volumes 70% ethanol.
4. Equilibrate with 2 or 3 column volumes of column equilibration buffer,
or 20% ethanol and store at 4ºC, if the column is not used for a long
period of time (more than a week). The colum may be reused 5-10
times.
d. The target protein may be eluted using lower choline concentrations
(from 30 mM choline), but the collected sample is more diluted.
e. Protein quantitation: C-LYTAG has an E 0.1%(280nm)=3.72 and a
MW=21287 Da.
13
3.3.5. Choline elimination
To eliminate the choline, dialyze against phosphate buffer 20 mM pH 7.0 plus
50 mM NaCl. The dialysis can be done at temperature range between 4ºC-room
temperature. When the sample is very concentrated choline elimination may cause
protein precipitation.
Once choline is removed, or its concentration is diluted below 10mM, the CLYTAG fusion protein is able to bind again to C-LYTRAP.
3.3.6. Enterokinase cleavage
12
C-LYTAG fusion proteins carry an enterokinase cleavage site that allows for
removal of the C-LYTAG moiety. Enterokinase is a specific protease that cleaves
at Asp-Asp-Asp-Asp-Lys- after the lysine residue. The amount of enzyme required
to cleave a fusion protein in a 16 h reaction at room temperature ranges from
0.001% to 0.5% (w/w). Depending on the particular fusion protein, the amount
of protease can be adjusted within this range. Follow your enterokinase
manufacturer´s instructions for an optimal cleavage of the fusion proteins.
3.2. Protein expression
Optimal expression conditions should be determined for each particular
recombinant protein with small scale cultures before attempting large scale
expression procedures. Expression can be optimized by varying inducer
concentration (0.5-5 mM salicylate), temperature (22-37ºC) and time of
induction (4 h-overnight).
1. Inoculate a pre-culture with a single, freshly transformed colony. Add
the appropriate antibiotics (25 mg/ml kanamycin and 100 mg/ml
ampicillin) to the pre-culture and incubate overnight at 37ºC with
shaking (225 r.p.m.).
2. Inoculate a culture with a 1:100 dilution of the pre-culture. Add the
appropriate antibiotics and grow the culture at 37ºC, 225 r.p.m. for
2-2.5 hours (until the OD600 is 0.2-0.3).
3. Add the expression inducer (salicylate) to a final concentration of 2
mM.
4. Grow induced culture for 5 h at 30ºC and 225 r.p.m.
5. Harvest the culture by centrifugation at 4000 x g for 15 min at 4°C.
Remove the supernatant and store the pellet at –20°C until needed.
3.3.7. Column regeneration and storage
If contaminants are not detected:
1. Wash adding 2-3 volumes of elution buffer.
2. Equilibrate with 5 column volumes of column equilibration buffer, or 20%
ethanol if the column is not used for a long period of time (more than a
week). Store at 4 ºC.
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3.3. Protein purification
Optimal purification conditions should be determined for each particular
recombinant protein with small scale cultures before attempting large scale
purification procedures.
C-LYTAG PURIFICATION SYSTEM
Choline is basically a non-reactive and optically transparent molecule, therefore
removal from the purified sample may not be necessary, thus avoiding additional
purification steps (desalting, dialysis, chromatography, etc.). Besides, C-LYTAG
is more stable when choline is present, although choline may induce dimerization
of the fusion protein via C-LYTAG (3) and it may be desirable to remove the eluent
in some cases.
- Transformation of E. coli REG-1 strain may be performed using standard
methods. Biomedal recommends TSS solution (Cat.No. RS-3215/16), a fast
and simple system to prepare competent cells (for information
www.biomedal.es).
9
The following protocol is recommended for cultures of 0.5-4 L. Volumes of
resin and buffers can be scaled in this range of culture volume. Conditions for
larger and smaller culture volumes must be optimized.
3.3.1. Column preparation
NOTE u a. During shipping and storage, the resin will settle. We recommend thoroughly
resuspending it before pipetting.
b.The column must not be allowed to run dry. If it run dry, resuspend the resin
in column equilibration buffer and repeat the packing.
3.3.2. Extract cell preparation
10
1. Resuspend the pellet (obtained in step 5 of Section 3.2) in 50 ml of cell
resuspension buffer per litre of culture. Keep the sample on ice and disrupt
the cells by sonication or using a French press.
2. Centrifuge at 9000 x g for 20 minutes to pellet the cell debris.
3. Remove the supernatant (crude extract) to a fresh container and save on ice.
4. Some proteins form inclusion bodies when they are expressed at high levels
in bacteria. At this point, it is important to determine whether the expressed
protein is soluble or is located in the insoluble fraction (inclusion bodies) by
means of SDS-PAGE or Western–blot. If the expressed protein is located in
the insoluble fraction, the pellet must be solubilized following the procedure
below: (see Section 4 for composition of buffer )
1. Resuspend the pellet (obtained in step 2) using 10 ml of Buffer 1.
2. Centrifuge at 9000 x g (10 min) and discard pellet.
3. Dialyze for 4 h against 500 ml of Buffer 2 at 20ºC.
4. Dialyze for 3 h against 500 ml of Buffer 3 at 20ºC.
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NOTE u Although C-LYTAG domain does not form inclusion bodies by itself, the hybrid
protein may do so. This protocol has been succesfully tested in many cases,
but it may need readjusting depending on each particular protein (addition
of detergents, variation of temperature, etc.). On the other hand, direct dialysis
of the resuspended precipitate from point 1 above against Buffer 4 may also
yield good results.
5. Load the crude extract or the solubilized inclusion bodies onto the C-LYTRAP
column. We recommend a flow rate of not higher than 1 ml/min in order
to allow a thorough contact between extract and resin.
3.3.3. Column washing and elution
1. Wash the column with 10 volumes of washing buffer or until OD280 is less
than 0.01.
2. Re-equilibrate with 2 volumes of re-equilibrating buffer.
3. Elute the fusion protein with 3-4 volumes of elution buffer. Collect fractions
and analyze them to detect the presence of the fusion protein by means of
colorimetric assay, measurement of OD280 or using anti C-LYTAG antibodies
Biomedal (Cat. No. AB-3238).
3.3.4. Batch purification
Proteins may be purified on C-LYTRAP resin in either a batch or a column
procedure. When the fusion protein is expressed at low levels or the crude
extract is viscous, protein yields can be improved using batch purification. This
procedure entails binding the protein to the C-LYTRAP resin in solution, mixing
the resin and the crude extract in a flask and shaking gently for at least 1 h.
The washing and elution may be carried out either in batch or in a column
procedure, as is described in Section 3.3.
C-LYTAG PURIFICATION SYSTEM
C-LYTRAP is supplied pre-swollen as a 75% slurry, stored in 20% ethanol.
Pack the required amount of resin (8 ml C-LYTRAP per litre of culture) in an
appropriate column and equilibrate with 2-3 volumes of column equilibration
buffer. Ensure that the equilibration buffer completely replaces the 20% ethanol
buffer.
5. Dilute 1:10 with 90 ml of Buffer 4.
6. Centrifuge at 9000 x g (10 min). The supernatant is ready to be
loaded onto the C-LYTRAP column.
11
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