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 µl elution buffer and check on 1% agarose gel load 2 µl. 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 37°C 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 bradford’s 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) 550–563. 8. Zachary I: “Vascular endothelial growth factor”. Int J Biochem Cell Biol 1998;30: 1169–1174.
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