PURIFICATOIN BY IMAC OF A SUB-CLONED AND EXPRESSED GFP
Aranza Lopez
17611854 Group B Friday
The green fluorescent protein (GFP) of the appropriated restriction endonuclease (Inoue et
jellyfish Aequorea Victoria is commonly used as al., 1990). The transformed cells were synthesized
a reporter molecule for localization and with hexahistidine-tagged GFP and then purified
expression (Tsien, 1998). To gain experience in by IMAC on nickel agarose (Hochuli et al., 1988).
current techniques in molecular and medical GFP purification was analyzed by SDS-PAGE and
cell biology, the open reading frame (ORF) of a then transferred to a NCF and analyzed by
GFP was sub-cloned into a bacterial expression Western blot using specific antibodies that
vector and then purified by immobilized metal targeted our GFP (Swerdlow et al, 1986). The
affinity chromatography (IMAC). At the end of purity and yield of our GFP was determined. The
this experiment, the purity and yield of our purity of the GFP in the crude lysate and the
GFP were quantitatively and qualitatively eluent was compared and expressed as a
determined. The purification following IMAC percentage of total protein. On the other hand, this
was only 2.4, which was low, compared to the experiment helped us understand the different and
potential purification for this procedure better techniques used to purify proteins.
reported by Hochuli et al. (1988). In addition to
the expression and purification of our
recombinant GFP, this experiment enabled us
to experience with a bread range of molecular EXPERIMENTAL PROCEDURES
biology techniques used in research
laboratories.
Amplification of the ORF using PCR and analysis
by Agarose Electrophoresis. Our GFP ORF was
INTRODUCTION amplified using PCR by adding: 24.25 µl of
sterile H2O, 5 µl 10x Vent buffer, 5 µl of pE-GFP
template (10ng/µl), 5 µl of primer E1 (5pmol/µl),
5 µl of primer E2 (5pmol/µl), 5 µl of 2.5 mM
Green fluorescent protein is a great tool in dNTPs and 0.75 µl of Vent polymerase. For 0.8%
molecular and medical biology; its fluorescent agarose electrophoresis analysis, the PCR product
properties are commonly used for localization and was diluted with 150 µl of sterile H2O and 5µl of
expression (Tsien, 1998). During this experiment, 6x DNA sample buffer. After amplification, DNA
the ORF of the GFP from a pE-GFP vector was was purified using Promega SV Mini-columns.
amplified by PCR (Barnes, 1992), to ensure that
our RCR result did not have mutations; we used Restriction digest of GFP and pQE30 vector for
Vent polymerase, which is a thermostable DNA ligation. 18 µl of GFP and pQE30 DNA were
polymerase that contains proofreading. GFP DNA prepared with 2 µl 10x buffer E and 5 µl
was purified and digested with appropriated BamHI/HindIII premix. Samples were digested
restriction enzymes (BamHI & HindIII) and then for 2 hours at 37oC. Digested GFP DNA insert and
ligated to a digested vector containing powerful the cut pQE30 vector were purified using SV
bacteria promoter and convenient hexahistidine- Mini-columns. After purification, ligation was
tag for protein purification. PCR and ligation achieved by mixing of 10.20 µl of nuclease free
results were analyzed by agarose electrophoresis water, 6.25 µl of digested pQE30 vector, 0.55 µl
(Laemmli, 1970). After ligation, competent E.coli of digested GFP DNA insert, 2 µl of 10x T4 DNA
cells were transformed with our new pQE30-GFP ligase buffer and 1µl of T4 DNA ligase.
construct, bacteria containing the new construct
was able to fluoresce under UV light and its Preparation of competent cells by Inoue method
identity was confirmed by digestion with and transformation with pQE30-GFP construct.
1
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https://www.coursehero.com/file/17395424/GFP-REPORT/
, 50 µl of chilled E.coli cells were centrifuged at density (OD600) of the suspension at the time of
2500g for 10 minutes at 4oC. Ice-cold Inoue the collection. For SDS-PAGE, Proteins were
transformation buffer (27ml) was slowly added denaturized by incubating them at 950C for two
and mixed into the E.coli cells. The mixture was minutes before SDS-PAGE analysis. The resulting
centrifuge at 2500g for 10minuts at 4 oC and the samples were separated by electrophoresis on
supernatant was discarded. Finally, the cells were 10% SDS-PAGE and electrophoresed at 120-150
resuspende with 2 ml of ice-cold Inoue V. Samples were stained in the gel by Comassie
transformation buffer and 150 µl of DMSO. Cells brilliant blue solution and distained by repeated
were sub-liquated, snap-freeze in N2 and washing in distaining solution (Laemmli, 1970).
incubated at -70oC. After Inoue preparation
method, the ligation sample was heat-incubated at GFP Western Blot analysis. Protein extracts were
65oC and then mixed with the competent cells. analyzed by 10% SDS-PAGE, transferred onto a
Before purification, transformation results were nitrocellulose filter, and GFP was detected using
analyzed, GFP cloning was confirmed by using polyclonal rabbit anti-GFP IgG diluted 1/1000 in
plasmid mini-prep and restriction enzyme and 1% (w/v) skim milk, tris-buffer saline. The
hexahistidine-tagged GFP induction was achieved secondary antibody was alkaline phosphate-
(Inoue et al. 1990). conjugated goat anti-rabbit IgG diluted 1/5000 in
1% (w/v) skim milk, tris-buffered saline.
Purification of hexahistidine-tagged GFP from
transformed cells using IMAC. Cell pellets from
about 40 ml of suspension with a density of 2.163
OD600 collected 2 hours post-induction were lysed RESULTS
with 5 ml of pre-measured lysis buffer (50 mM
NaH2PO4, pH 8.0; 300 mM NaCl; plus 0.2 mg/ml
lysozyme added fresh). The cells were
resuspended and incubated for 20 minutes in the Amplified GFP ORF by PCR. In order to
370C water bath, after incubation, magnesium determine the purity and yield of our sub-cloned
chloride and deoxyribonuclease was added. For and expressed GFP, we first amplified the GFP
10 minutes the cells were incubated at room ORF from a pE-GFP vector by PCR. The PCR
temperature and then sonicated. product was visualized by agarose electrophoresis
and demonstrated in figure1. Three bands of about
The crude cell homogenate was centrifuged at full 750bp were detected (lanes 3 - student 1, 5 -
speed for 1 minute and then mixed with 100 µl of student 2 and 6), one of them being the positive
5% slurry of Nickel-nitrilotriacetic acid (NI-NTA) control (lane 6). An unexpected extra band cause
agarose beads in lysis buffer (50% w/v beads in by RCP product sample excess was also detected
lysis buffer), gently shaken for one hour at room (lane 4 - student 1). Compering with the control
temperature and centrifuge for 1 minute at 226.4g. band in lane 6, bands in lane 3 and 5, appear to
The pellet was subsequently washed 4 times with have the right size and sufficient product (Barnes,
1 ml of wash buffer (50mM NaH2PO4, pH 8.0; 1992).
300mM NaCl) for 5 minutes and then centrifuged
for 1 minute at 226.4g; supernatant was not DNA digests by Agarose Electrophoresis. The
collected after the second and third wash. To elute DNA from GFP and the cut pQE30 vector were
the proteins, the wash beads were resuspended digested with BamHI and HindIII restriction
with 250 µl of elution buffer (50mM NaH2PO4; enzymes and separated by agarose electrophoresis
pH 8.0; 300mM NaCl; 250mM imidazole), (figure 2). Four bands from 2 different students
incubated for 5 minutes at room temperature and can be visualized. The estimated size in bands
then centrifuge for 1 minute at 226.4g. After each from lanes 7 and 4 is 4,000bp and lanes 6 and 2 is
centrifugation, the supernatant was collected for 750bp. Bands in lane 6,4 and 3 seem to be clear
SDS-PAGE analysis (Hochuli et al., 1988). and bright, but in the other hand, band in lane 7
cannot be seen clearly.
Preparing the induction and GFP purification
sample for SDA-PAGE analysis. Prior to SDS- Transformed competent E.coli cells with pQE30-
PAGE, induction time samples were treated with GFP construct. Plasmid DNA from transformed
proportional volumes of water and intensely blue cells was isolated and its identity was confirmed
sample-loading buffer depending on the cell by digestion with appropriated restriction
2
This study source was downloaded by 100000850872992 from CourseHero.com on 02-20-2023 00:26:59 GMT -06:00
https://www.coursehero.com/file/17395424/GFP-REPORT/
Aranza Lopez
17611854 Group B Friday
The green fluorescent protein (GFP) of the appropriated restriction endonuclease (Inoue et
jellyfish Aequorea Victoria is commonly used as al., 1990). The transformed cells were synthesized
a reporter molecule for localization and with hexahistidine-tagged GFP and then purified
expression (Tsien, 1998). To gain experience in by IMAC on nickel agarose (Hochuli et al., 1988).
current techniques in molecular and medical GFP purification was analyzed by SDS-PAGE and
cell biology, the open reading frame (ORF) of a then transferred to a NCF and analyzed by
GFP was sub-cloned into a bacterial expression Western blot using specific antibodies that
vector and then purified by immobilized metal targeted our GFP (Swerdlow et al, 1986). The
affinity chromatography (IMAC). At the end of purity and yield of our GFP was determined. The
this experiment, the purity and yield of our purity of the GFP in the crude lysate and the
GFP were quantitatively and qualitatively eluent was compared and expressed as a
determined. The purification following IMAC percentage of total protein. On the other hand, this
was only 2.4, which was low, compared to the experiment helped us understand the different and
potential purification for this procedure better techniques used to purify proteins.
reported by Hochuli et al. (1988). In addition to
the expression and purification of our
recombinant GFP, this experiment enabled us
to experience with a bread range of molecular EXPERIMENTAL PROCEDURES
biology techniques used in research
laboratories.
Amplification of the ORF using PCR and analysis
by Agarose Electrophoresis. Our GFP ORF was
INTRODUCTION amplified using PCR by adding: 24.25 µl of
sterile H2O, 5 µl 10x Vent buffer, 5 µl of pE-GFP
template (10ng/µl), 5 µl of primer E1 (5pmol/µl),
5 µl of primer E2 (5pmol/µl), 5 µl of 2.5 mM
Green fluorescent protein is a great tool in dNTPs and 0.75 µl of Vent polymerase. For 0.8%
molecular and medical biology; its fluorescent agarose electrophoresis analysis, the PCR product
properties are commonly used for localization and was diluted with 150 µl of sterile H2O and 5µl of
expression (Tsien, 1998). During this experiment, 6x DNA sample buffer. After amplification, DNA
the ORF of the GFP from a pE-GFP vector was was purified using Promega SV Mini-columns.
amplified by PCR (Barnes, 1992), to ensure that
our RCR result did not have mutations; we used Restriction digest of GFP and pQE30 vector for
Vent polymerase, which is a thermostable DNA ligation. 18 µl of GFP and pQE30 DNA were
polymerase that contains proofreading. GFP DNA prepared with 2 µl 10x buffer E and 5 µl
was purified and digested with appropriated BamHI/HindIII premix. Samples were digested
restriction enzymes (BamHI & HindIII) and then for 2 hours at 37oC. Digested GFP DNA insert and
ligated to a digested vector containing powerful the cut pQE30 vector were purified using SV
bacteria promoter and convenient hexahistidine- Mini-columns. After purification, ligation was
tag for protein purification. PCR and ligation achieved by mixing of 10.20 µl of nuclease free
results were analyzed by agarose electrophoresis water, 6.25 µl of digested pQE30 vector, 0.55 µl
(Laemmli, 1970). After ligation, competent E.coli of digested GFP DNA insert, 2 µl of 10x T4 DNA
cells were transformed with our new pQE30-GFP ligase buffer and 1µl of T4 DNA ligase.
construct, bacteria containing the new construct
was able to fluoresce under UV light and its Preparation of competent cells by Inoue method
identity was confirmed by digestion with and transformation with pQE30-GFP construct.
1
This study source was downloaded by 100000850872992 from CourseHero.com on 02-20-2023 00:26:59 GMT -06:00
https://www.coursehero.com/file/17395424/GFP-REPORT/
, 50 µl of chilled E.coli cells were centrifuged at density (OD600) of the suspension at the time of
2500g for 10 minutes at 4oC. Ice-cold Inoue the collection. For SDS-PAGE, Proteins were
transformation buffer (27ml) was slowly added denaturized by incubating them at 950C for two
and mixed into the E.coli cells. The mixture was minutes before SDS-PAGE analysis. The resulting
centrifuge at 2500g for 10minuts at 4 oC and the samples were separated by electrophoresis on
supernatant was discarded. Finally, the cells were 10% SDS-PAGE and electrophoresed at 120-150
resuspende with 2 ml of ice-cold Inoue V. Samples were stained in the gel by Comassie
transformation buffer and 150 µl of DMSO. Cells brilliant blue solution and distained by repeated
were sub-liquated, snap-freeze in N2 and washing in distaining solution (Laemmli, 1970).
incubated at -70oC. After Inoue preparation
method, the ligation sample was heat-incubated at GFP Western Blot analysis. Protein extracts were
65oC and then mixed with the competent cells. analyzed by 10% SDS-PAGE, transferred onto a
Before purification, transformation results were nitrocellulose filter, and GFP was detected using
analyzed, GFP cloning was confirmed by using polyclonal rabbit anti-GFP IgG diluted 1/1000 in
plasmid mini-prep and restriction enzyme and 1% (w/v) skim milk, tris-buffer saline. The
hexahistidine-tagged GFP induction was achieved secondary antibody was alkaline phosphate-
(Inoue et al. 1990). conjugated goat anti-rabbit IgG diluted 1/5000 in
1% (w/v) skim milk, tris-buffered saline.
Purification of hexahistidine-tagged GFP from
transformed cells using IMAC. Cell pellets from
about 40 ml of suspension with a density of 2.163
OD600 collected 2 hours post-induction were lysed RESULTS
with 5 ml of pre-measured lysis buffer (50 mM
NaH2PO4, pH 8.0; 300 mM NaCl; plus 0.2 mg/ml
lysozyme added fresh). The cells were
resuspended and incubated for 20 minutes in the Amplified GFP ORF by PCR. In order to
370C water bath, after incubation, magnesium determine the purity and yield of our sub-cloned
chloride and deoxyribonuclease was added. For and expressed GFP, we first amplified the GFP
10 minutes the cells were incubated at room ORF from a pE-GFP vector by PCR. The PCR
temperature and then sonicated. product was visualized by agarose electrophoresis
and demonstrated in figure1. Three bands of about
The crude cell homogenate was centrifuged at full 750bp were detected (lanes 3 - student 1, 5 -
speed for 1 minute and then mixed with 100 µl of student 2 and 6), one of them being the positive
5% slurry of Nickel-nitrilotriacetic acid (NI-NTA) control (lane 6). An unexpected extra band cause
agarose beads in lysis buffer (50% w/v beads in by RCP product sample excess was also detected
lysis buffer), gently shaken for one hour at room (lane 4 - student 1). Compering with the control
temperature and centrifuge for 1 minute at 226.4g. band in lane 6, bands in lane 3 and 5, appear to
The pellet was subsequently washed 4 times with have the right size and sufficient product (Barnes,
1 ml of wash buffer (50mM NaH2PO4, pH 8.0; 1992).
300mM NaCl) for 5 minutes and then centrifuged
for 1 minute at 226.4g; supernatant was not DNA digests by Agarose Electrophoresis. The
collected after the second and third wash. To elute DNA from GFP and the cut pQE30 vector were
the proteins, the wash beads were resuspended digested with BamHI and HindIII restriction
with 250 µl of elution buffer (50mM NaH2PO4; enzymes and separated by agarose electrophoresis
pH 8.0; 300mM NaCl; 250mM imidazole), (figure 2). Four bands from 2 different students
incubated for 5 minutes at room temperature and can be visualized. The estimated size in bands
then centrifuge for 1 minute at 226.4g. After each from lanes 7 and 4 is 4,000bp and lanes 6 and 2 is
centrifugation, the supernatant was collected for 750bp. Bands in lane 6,4 and 3 seem to be clear
SDS-PAGE analysis (Hochuli et al., 1988). and bright, but in the other hand, band in lane 7
cannot be seen clearly.
Preparing the induction and GFP purification
sample for SDA-PAGE analysis. Prior to SDS- Transformed competent E.coli cells with pQE30-
PAGE, induction time samples were treated with GFP construct. Plasmid DNA from transformed
proportional volumes of water and intensely blue cells was isolated and its identity was confirmed
sample-loading buffer depending on the cell by digestion with appropriated restriction
2
This study source was downloaded by 100000850872992 from CourseHero.com on 02-20-2023 00:26:59 GMT -06:00
https://www.coursehero.com/file/17395424/GFP-REPORT/
