Molecular cloning of Green fluorescent protein (GFP) and protein
purification via PCR, IMAC in bacteria E coli cells.
Received for publication, 04 June 2021
In this experiment, Green fluorescent protein
(GFP) was clone and purified. To start the
molecular cloning of GFP, pE-GFP plasmid that
holds in GFP open reading frame used in
polymerase chain reaction for DNA sequence
amplification. The bacterial expression vector that
holds in the hexahistidine-tag, pQE-30 vector used.
The aim was to amplify the GFP DNA sequence
(insert) from the vector using specific primers and
prepare GFP DNA for the ligation reaction with
pQE30 vector to form pQE30+GFP plasmid. For
ligation reaction to take place both vector and
insert were digested using restriction enzyme
digest BamHi and HindIII which were purified
using Promega SV Mini-columns Wizard DNA
purification kit. The competent E. Coli XL 1-Blue
cells and ligated DNA transformed in agar plates.
Incubated for appropriate amount of time and
GFP induction were done. The Wizard DNA
purification kit used to purify the GFP from the
bacteria cells. Finally, GFP expressed purified
from all other E. Coli proteins via Immobilized
Metal Affinity Chromatography (IMAC).
Introduction
Scientific and medicinal research is based on studying
proteins analysing its structure and function. One
major use of proteins is for producing anti-bodies for
diagnostic purposes (Young et al. 2012). Protein
purification and protein localisation in cells are two
important aspects necessary to consider in the
research field. Protein localisation in cells is achieved
via using Green fluorescent protein (GFP), a naturally
occurring fluorescent protein which is found in
jellyfish. It consists of 11 anti-barrel β strands
threaded by an α-helix in its centre, the α-helix
contains three critical residues Serine, Tyrosine and
glycine that are important for absorbing and emitting
light (Dantuma et al. 2000). This is a substantial
method used in molecular cloning and protein
expression in order to visualise protein activity in the
cell.
In this experiment we aimed to clone, express and
purify GFP using molecular biology techniques.
Molecular cloning of GFP was done via using a
plasmid pE-GFP, that contains GFP open reading
frame. Because the aim was to express the
recombinant protein in bacteria E. Coli cells the pEQ30 vector which is designed for bacteria expression
was used. This vector also contains ampicillin
resistance marker hexahistidine-tag (6his-tag) which
allows for selective protein purification using
Immobilized Metal Affinity Chromatography
(IMAC) technique.
Material and methods
There were five parts in this experiment, number of
steps covered in each part. The Initial part of the
experiment designed to achieve PCR, Purification and
Agarose Gel Electrophoresis of DNA encoding GFP
from the pE-GFP vector.
The PCR amplification of GFP DNA conducted using
10x Vet buffer, pE-GFP templet, E1 and E2 primers.
Before the start of PCR an initial denaturation of DNA
was done at 94 °C for 2 minutes. Next the PCR
reaction began for 30 cycles, denaturation at 94 °C for
30 sec, annealing at 50 °C for 30 sec, extension at 72
°C for 45 sec, final extension at 72 °C for 3 min which
was hold at the end till the reaction reached 4 °C.
Agarose gel electrophoresis (0.8% w/v) in TBE buffer
used to analyse the PCR results (Figure, 1). The GFP
DNA clone was purified using Promega SV minicolumns, Wizard DNA purification kit.
In the second part of the experiment aimed to
add/insert the clone GFP into the pQE30 expression
vector. First, pQE-30 (vector) and pE-GFP (insert)
were digested using restriction enzymes (ER) BamHI
and HindIII in 10x buffer E. The Nanodrop 2000
spectrophotometer was used to check the purity of the
solution before the ligation reaction conducted
(Table, 1). Ligation reaction was done using purified
digested insert and vector, T4 DNA ligase. The results
analysed via agarose gel electrophoresis (0.8 %w/v),
(Figure, 2).
Table 1. Nanodrop 2000 spectrophotometer results and
calculated concentration of both vector and insert.
In the third part of the experiment competent E.Coli
cells prepared and transformation of bacteria with
ligation products conducted. E Coli. XL 1-Blue cells
prepared with OD600 of 0.55 with Inoue
Transformation buffer and DMSO. This followed by
transformation of E. Coli cells with ligated DNA in
LB agar that contained 600 µL of ampicillin and 150
µL of IPTG. The plated incubated overnight at 37 °C
(Table, 2).
Table 2. Bacterial transformation in agar plates samples.
After sufficient period of time the agar plates colonies
with GFP in the E. Coli cells examined and the GFP
induction was done.
The pQE30-GFP and pQE30 cultures were inoculated
and allowed to grow cultures overnight in Terrific
broth that contained ampicillin. The plasmid DNA
prepared via miniprep technique Wizard DNA
purification kit and was digested using BamHI and
HindIII restriction enzymes. The results analysed by
gel electrophoresis (0.8% w/v) that contain SYBR
Safe dye and TBE buffer and 6x DNA loading dye
(Figure, 3)
In the final step the cloned GFP protein was induced
via lysis buffer and purified via IMAC. The results
analysed via 10% SDS-PAGE electrophoresis
(Figure, 4).
Results and Discussion
In molecular biology recombinant DNA and
recombinant protein expression are the tool used in
research filed. Protein cloning and purification is used
in biomedical studies hence, it is important to be able
to localise expressed protein in the cells. This can
achieve via Green fluorescent protein (GFP). This
protein contains amino acid that can absorb and emit
light. This experiment aimed to clone and purify
expressed GFP via molecular biology techniques
PCR, IMAC, Promega SV mini-columns Wizard
DNA purification kit. There were a total of five steps
in this experiment, each part correlated to next.
The GFP DNA sequence amplified from the vector
pE-GFP via PCR. The amplification conducted using
E1 and E2 primers. GFP opening reading frame is 720
bp and its digested GFP is 741 bp this was absorbed
in the PCR conducted lane 1 in Figure 1. The control
sample used in lane 7 in Figure 1 concurred this result.
Bacteria cells are largely used to express proteins, this
is a cheap and large amount of protein can be
conducted (Hyung Joon 2005). The vector pE-GFP is
not appropriate for bacteria expression, hence pQE30
was used that also contains ampicillin resistance
marker (6 His-tag). The hexahistidine-tag allows for
better selection and provides pure recombinant
protein during the purification step.
For the ligation reaction step; RE digestion are require
in ligation reaction to join the insert and vector via
producing sticky ends in this case. It is important that
RE sequence are not present in the insert and vector.
Hence the ER used in this experiment were BamHI
and HindIII, these two ER are requiring the same
condition which is another advantage (Jia & Jeon
2016). In order to avoid star activity a premix solution
of ER was used that contained <10% of the ER and
5% (V/V) of glycerol (New England Biolabs, 2012).
PCR results for Digested insert and vector was as
expected. The digested pQE30 vector with 3419 bp
appeared between 5000 – 2000 bp. GFP insert with
741 bp showed a band just below the 850 bp, refer to
figure 2 lane 6 and 7 respectively. The agarose gel
electrophoresis used 0.8% (w/v) this was to ensure
that the sample would not run off the agarose page.
The results from cell transformation for digested
pQE30+GFP was as expected. The Green Fluorescent
colonies were about 40 and 176 of cream colonies.
Possibly this could due to re-ligation of vector with
itself as the ligation did not take place. The results for
digested pQE30 contain 1.3 Green Fluorescent
colonies, this might be due contamination and 172
cream colonies as it was expected (Table 2).
From the agar plates that contained pQE30+GFP and
pQE30 colonies plasmid DNA were prepared. This
was done by inoculating the colonies and allow for
overnight culture to grow. Where the Wizard DNA
purification kit use and was digested used both
BamHI and HindIII ER. To analyse the results both
pQE30+GFP and pQE30 sample conducted as cut and
undigested. Figure 3, lane 1 contain uncut
pQE30+GFP that run through the gel and have a few
bands which is due to supercoiled and linear DNA
(Min et al. 2005). In lane 2 contain digested
pQE30+GFP that has two band which is due to insert
GFP DNA and vector. Lane 3 contains uncut pQE30
that has two bands due to supercoiled and linear DNA
and lane 4 is the digested pQE30 that had only one
band between 5000 – 2000 bp.
Purification of induced GFP
Before the start of protein purification, the two
cultures incubated in LB broth that contained
ampicillin to ensure only GFP to grow and IPTG to
help with protein growth in the cells. To ensure the
accuracy and to be able to analyse of the results
OD600 value for zero hour before the induction,
+1hour induction and +2hour induction measured
(Table, 3). This is the optimal density at 600
wavelengths for turbidity of culture (Jia & Jeon 2016).
Sample
Pre-induction (0)
1 hour post induction (+1)
2 hour post induction (+2)
OD600
0.773
1.170
1.780
In order to extract GFP from all other proteins that are
present in the E. Colo cells lysis buffer used. This was
to release the GFP that contains his-tag into solution
this was due to use of Lysozyme that can cleave
peptidoglycan which lead to breaking the cell wall
(the Crude cell extract) this was illustrated in the SDSPAGE result figure 4, lane 5 (Young et al. 2012).
The crude cell extract sample used in protein
purification via IMAC system, to pellet the GFP out
from the bacterial cells which was then purified in
number of steps in the final of step larger amount of
imidazole used to release His-tagged GFP. Because
imidazole have similar structure to histidine it can be
used to elute His-tagged GFP from the NI-NTA beads
(Min et al. 2005). Figure 4 is the results illustrating
that His-tagged GFP was purified which is in lane 2
the band is located between 38 – 28 kDa which as it
was expected 27.5 kDa.
The results illustrate high skills of molecular biology
used which was to prevent any contamination hence,
to produce purify protein at the end. Because protein
purification is essential tool in molecular biology
laboratory different protein purification system could
be used to confirm the results. This could be using the
ProtParam program to look up number of amino acids
to be able to calculate the molecular weight of the
protein. This allows us to separate proteins basis of
molecular size via gel filtration chromatography.
.
References
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Young, Carissa L, Britton, Zachary T & Robinson,
Anne S, 2012. Recombinant protein expression and
purification: A comprehensive review of affinity tags
and microbial applications. Biotechnology journal.,
7(5), pp.620–634.
Min, L., Xing-guo, G., Hong, Y. and Jian-yong, L.,
2005. Cloning, expression, purification, and
characterization of LC-1 ScFv with GFP tag. Journal
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(n.d.).
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Restriction Endonuclease Reactions | NEB. [online]
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at:
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Figures and figure legends
Part 1 of the experiment results. Polymerase chain reaction (PCR), purification and agarose
gel electrophoresis.
Figure 1. The agarose gel (0.8% w/v) of PCR product. The PCR amplification of GFP DNA sequence from the pE-GFP
vector using specific primers. Which was ≈ 720 bp. Illustrated in lane 1 and 2 respectively.
Second part, cloning of GFP into the expression vector pQE30.
Figure 2. The agarose gel (0.8% w/v) of PCR products of digested inset and vector. Digestion of GFP DNA and pQE30
vector preformed with BamHI and HindIII. Lane 6 and 7 illustrate the results respectively.
The PCR results both pQE30+GFP and pQE30 sample conducted as cut and undigested.
Figure 3. The plasmid DNA restriction digests showing in agarose gel (0.8% w/v) containing SYBR safe dye. Lane 2 is
representing the cut pQE30-GFP.
The SDS-PAGE results for IMAC purification of His-tagged GFP. .
Figure 4. The 10% SDS-PAGE electrophoretic analysis of GFP fusion protein purification using IMAC. The final purified
GFP presented in lane 2 it is located at ≈ 28 kDa.
The table below is the DNA ladder used in this experiment for the electrophoresis.
Table 3. DNA markers ladder.
DNA
Ladder
used for PCR gel
electrophoresis
DNA
Ladder
used for SDSPAGE
electrophoresis