IJBSTR RESEARCH PAPER VOL 1 [ISSUE 2] FEBRUARY 2013

ISSN 2320-6020

CLONING, EXPRESSION AND PURIFICATION OF MOUSE

VEGF (Vascular Endothelial Growth Factor) in E. coli
Ashish Kumar Chaudhary and Sandeep Vishwakarma

ABSTRACT- Vascular endothelial growth factor (VEGF) is a chemical signal produced by cells that stimulates the growth of new
blood vessels and restores the oxygen supply to tissues when blood circulation is inadequate. The most important member is VEGF-A.
Other members are Placenta growth factor (PlGF), VEGF-B, VEGF-C and VEGF-D. VEGF expression is normally low in skin
relative to other more highly vascularized organs such as lung, kidney, and heart. VEGF was isolated from culturing mouse cells. The
VEGF gene was identified after sequencing of the PCR product. VEGF of length 498bp was isolated from cultured mouse cell and
cloned into BL21 (DE3) expression strain of E. coli cells. The molecular weight of the protein was determined to be 18.26 KDa from
12% SDS PAGE. The protein was successfully purified after expression using Ni-NTA matrix.

Key words: Vascular endothelial growth factor (VEGF), VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF receptors.
INTRODUCTION
Vascular endothelial growth factor (VEGF), a
dimeric 42-kd protein, is a multifunctional cytokine
that plays a pivotal role in angiogenesis (Ferrara N,
1999). VEGF is a key regulator of physiological
angiogenesis during embryogenesis, skeletal growth
and reproductive functions. VEGF has also been
implicated in pathological angiogenesis associated
with tumors, intraocular neovascular disorders and
other conditions. The biological effects of VEGF are
mediated by two receptor tyrosine kinases (RTKs),
VEGFR-1 and VEGFR-2, which differ considerably
in signaling properties (Ferrara N, 1999). VEGF's
normal function is to create new blood vessels during
embryonic development, new blood vessels after
injury, muscle following exercise, and new vessels
(collateral circulation) to bypass blocked vessels.
When VEGF is over
expressed, it can contribute to disease. Solid cancers
cannot grow beyond a limited size without an
adequate blood supply; cancers that can express
VEGF are able to grow and metastasize.

Author:
Ashish
Kumar
Chaudhari
Department of Biotechnology, Lovely
Professional University, Phagwara, Punjab.
E-mail: achaudhary2009@gmail.com

Co-Author: Sandeep Vishwakarma

Over expression of VEGF can cause vascular disease

in the retina of the eye and other parts of the body.
Drugs such as bevacizumab can inhibit VEGF and
control or slow those diseases (Holmes K et al,
2007).
VEGF is a sub-family of growth factors,
specifically the platelet-derived growth factor family
of cystine-knot growth factors. They are important
signaling proteins involved in both vasculogenesis
(the de novo formation of the embryonic circulatory
system) and angiogenesis (the growth of blood
vessels from pre-existing vasculature) (Haigh J J).
The most important member is
VEGF-A. Other members are Placenta growth factor
(PlGF), VEGF-B, VEGF-C and VEGF-D (Tammela
T, 2005). Activity of VEGF-A as its name implies,
has been studied mostly on cells of the vascular
endothelium, although it does have effects on a
number of other cell types (e.g., stimulation
monocyte, macrophage migration, neurons, cancer
cells, kidney epithelial cells) (Greenberg et al,
2001). VEGF regulates several endothelial cell
functions, including mitogenesis, permeability,
vascular tone, and the production of vasoactive
molecules (Zachary I, 1998). In vitro, VEGF-A has

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IJBSTR RESEARCH PAPER VOL 1 [ISSUE 2] FEBRUARY 2013

been shown to stimulate endothelial cell mitogenesis

and cell migration. VEGF-A is also a vasodilator and
increases micro vascular permeability and was
originally referred to as vascular permeability factor.

ISSSN 2320-6020

VEGF gene cloned in the pTNOT cloning vector 3450 bp in size which has the
Bam H1\Xho1 site.

Materials and Methods

Cellular RNA was isolated from mouse cell culture
using TRIZOL method for RNA isolation. After
RNA isolation perform PCR for the DNA product.
The PCR is used to amplify a precise fragment of
DNA from a complex mixture of starting material
usually termed a template DNA and in many cases
requires little DNA purification. PCR consist of three
defined sets of times and temperature, termed steps
denaturation, annealing and extension. The bases
(complementary to the template) are coupled to the
primers on the 3' side (the polymerase adds dNTPs
from 5' to 3', reading the template from 3' to 5' side;
bases are added complementary to the template. The
cDNA produced by RT PCR was used as a template
for amplification of VEGF gene. Through PCR
analysis it is clear that VEGF gene was amplified
successfully from cDNA as it shows at 498bp on
1.5% agarose gel.

Figure 2: Map of pT-NOT cloning vector with Bam

H1 and Xho 1 site
In a digestion reaction VEGF gene and pT-NOT
vector both are digested with Bam H1 and Xho1
restriction enzyme for the same free end. The eluted
gene was ligated into T tailed cloning vector.
Ligase enzyme which ligates DNA fragments having
blunt, overhanging or complementary ends. In
ligation reaction, ratio of the plasmid and insert
should be in the balance. If the plasmid ratio is high
as compared to the insert then excess empty mono
and polymeric plasmid will be generated. If the ratio
is to low then the result may be an excess of linear
and circular homo and heteropolymers. After ligation
a reaction set up for the conformation to check
ligation are success are not. A restriction digestion of
these plasmids (pTNOT/VEGF) with same enzyme
Bam H1\Xho1 confirmed the presence of clone as it
released approx 498bp band on gel.

Figure 1: Confirmed VEGF Gene on agrose gel with

ladder
After conformation the size of the gene with respect
to ladder perform gel elution techniques. Through gel
elution eluted the fragment of VEGF. Eluted
fragment was also visible on gel, thus according to its
concentration it was used for ligation reaction.

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IJBSTR RESEARCH PAPER VOL 1 [ISSUE 2] FEBRUARY 2013

ISSN 2320-6020

(pET28a\VEGF) isolation dissolved pellet in 30 l

elution buffer and check on 1% agarose gel load 2 l.
Plasmid seen in 2,3,6 and 10 number of wells.

Figure 4: Clone (pTNOT/VEGF) are digested with

Bam H1\Xho1 which release approx same size gene.
After ligated plasmid (pTNOT / VEGF) are
transformed into E.coli strain TOP10 using the CaCl2
procedure. Usually this technique is used to introduce
a foreign plasmid into bacterial cell and to use those
bacteria to replicate the plasmid in order to make
large quantities of it. This is based on the natural
function of a plasmid to transfer the genetic
information vital to the survival of the bacteria.
VEGF gene obtained in multiple numbers of copies
with pT-NOT vector in E.Coli.
Now VEGF gene clone in pET28a vector which are
expression vector. The pET28a vector was digested
with Bam HI and Xho I. Digested by same restriction
enzyme VEGF gene inserted in pET28a and
subsequently transformed into E.coli strain TOP10
using the CaCl2 procedure.
Now to conformation the transformation of plasmid
in E.coli perform plasmid digestion with same
restriction enzyme after isolation of plasmid.
Isolation of plasmid DNA (pET28a\VEGF) was
performed using alkaline lysis protocol from the o/n
grown colonies in LB-Kanamycin. The plasmids
were digested with BamHI/XhoI to confirm the clone
and check the release of gene. After Plasmid

Figure 5: Plasmid (pET28a\VEGF) released gene

and plasmid fragment after digestion by BamHI/XhoI
The plasmid isolated from the transformed colonies
was transformed into BL21 (DE3). The expression
strain, BL21(DE3) containing the gene coding for the
protein of interest (VEGF) was inoculated in 50ml of
L.B-Kan+ media and incubated at 37C for 2hrs upto
1O.D. and collect pellet before induction and after
induction on different hours.
Collect pellet before induction and add 1M IPTG and
collect pellets 3hours\6hours\over night and check on
SDS PAGE.
Protein expressions are found good in 3hrs, 6hrs and
overnight after gel observation. The overnight IPTG
inducted sample was taken in falcon tube and
centrifuged at 5000rpm for 20mins. The pellet
obtained was dissolved in 20mM Tris. To the
dissolved pellet lysozyme was added and incubated at
room temperature for 2-3hrs. The cells were lysed
using sonication method. The sonicated sample was
centrifuged at 5000rpm for 20mins. The supernatant
was collected stored at 4oC after adding immidazole.

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IJBSTR RESEARCH PAPE VOL 1 [ISSUE 2] FEBRUARY 2013

The supernatant was applied to the Ni-NTA

resin and purification was performed as per the
standard defined protocol. The elutants were
collected at regular intervals and bradfords reagent
was added to test the presence of protein. The
elutants with positive results were run on 12% SDSPAGE to check the elution of the VEGF protein
(18.26 KDa).

ISSN 2320-6020

References
1. Ferrara N: Molecular and biological properties of
vascular endothelial growth factor. Journal of
Molecular Medicine1999, 77:527-543.
2. Greenberg J, Shields D: A role for VEGF as a
negative regulator of pericyte function and vessel
maturation. Section of Molecular Biology 2001.
3. Geniez MS, Arindel S. R: Endogenous VEGF Is
Required for Visual Function and Evidence for a
Survival
Role
on
Muller
Cells
and
Photoreceptors, November 2008 , Volume 3
4. Haigh J J, Role of VEGF in organogenesis.
Vascular Cell Biology Unit, Department for
Molecular Biomedical Research, Ghent University,
Ghent Belgium.
5. Holmes K, Roberts OL, Thomas AM, Cross MJ:
"Vascular endothelial growth factor receptor-2:
structure, function, intracellular signalling and
therapeutic inhibition" Cell Signal 19 (10) Oct
2007.

Figure 5: Elution proteins which are 18.26 KDa

Conclusion
VEGF was isolated from culturing mouse cells. The
VEGF gene was identified after sequencing of the
PCR product.
VEGF of length 498bp was isolated from cultured
mouse cell and cloned into BL21 (DE3) expression
strain of E. coli cells. The molecular weight of the
protein was determined to be 18.26KDa from
12%SDS PAGE. The protein was successfully
purified after expression using Ni-NTA matrix.
Expression of VEGF in E. coli so a properties to
develop to expression in the mammalian expression
vector. It helps in the treatment of disease involves
the use of cells to introduce gene coding therapeutic
proteins into the body.

6. Lee HJ, Kim KS, Park IH, Kim SU (2007): Human

Neural Stem Cells Over-Expressing VEGF
Provide Neuroprotection, Angiogenesis and
Functional Recovery in Mouse Stroke Model
PLoS ONE 2(1): (2) 1371
7. Tammela T, Enholm B, Alitalo K, Paavonen K: The
biology of vascular endothelial growth factors.
Cardiovascular Research 65 (2005) 550563.
8. Zachary I: Vascular endothelial growth factor.
Int J Biochem Cell Biol 1998;30: 11691174.

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