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Recombinant Human Epidermal Growth Factor (EGF)
(Cat. No.: C029)
Background:
Epidermal growth factor (EGF) is a small mitogenic protein that is thought to be involved in mechanisms such as normal cell growth, oncogenesis, and wound healing. This protein shows both strong sequential and functional homology with human type-alpha transforming growth factor (hTGF alpha), which is a competitor for EGF receptor sites. EGF is a small 53 amino acid residue long protein that contains three disulfide briges.
Description:
Epidermal Growth Factor (EGF) is a polypeptide growth factor which stimulates the proliferation of a wide range of epidermal and epithelial cells. Human EGF is a 6.2 kDa protein containing 53 amino acid residues.
Quality Control:
Biological activity: The ED50, calculated by the dose-dependant proliferation of murine BALB/c 3T3 cells is less then 2 ng/ml, corresponding to a specific activity of 5.0x 105 IU/ mg.
Purity: Greater than 95% as determined by
(a) Analysis by SEC-HPLC.
(b) Analysis by reducing and non-reducing SDS-PAGE Silver Stained gel.
Molecular weight: 6.0KD+/-10% determined by reduced SDS-PAGE.
Isoelectric Point: the main zone between 4.0~5.0 analysis by IEF.
UV scan: the maximal absorption wave is 275+/-3nm.
Amino-Acid Sequence: The sequence of the first fifteen N-terminal amino acids was determined and was found to be Asn-Ser-Asp-Ser-Glu-Cys-Pro-Leu-Ser-His-Asp-Gly-Tyr- Cys-Leu.
Residual DNA: less than 1ng/ug analysis by solid phase blot.
Residual host cell protein: less than 0.1% analysis by ELISA.
Endotoxin: Less than 0.3ng/µg (0.3IEU/µg) determined by LAL test.
Formulation: The protein was lyophilized from a concentrated (1mg/ml) solution with no additives.
Storage: Lyophilized rHuEGF although stable at room temperature for 3 weeks, should be stored desiccated below -18oC. Upon reconstitution rHuEGF should be stored at 4oC between 2-7 days and for future use below -18oC. For long-term storage it is recommended to add a carrier protein (0.1% HSA or BSA).
Please avoid freeze-thaw cycles.
Reconstitution: It is recommended to reconstitute the lyophilized rHuEGF in sterile 18MΩ-cm H2O not less than 100µg/ml, which can then be further diluted to other aqueous solutions.
Latest Publications:
1: Dev Biol 2008 Dec;
Wnt and EGF pathways act together to induce C. elegans male hook development.
[Abstract] Comparative studies of vulva development between Caenorhabditis elegans and other nematode species have provided some insight into the evolution of patterning networks. However, molecular genetic details are available only in C. elegans and Pristionchus pacificus. To extend our knowledge on the evolution of patterning networks, we studied the C. elegans male hook competence group (HCG), an equivalence group that has similar developmental origins to the vulval precursor cells (VPCs), which generate the vulva in the hermaphrodite. Similar to VPC fate specification, each HCG cell adopts one of three fates (1 degrees , 2 degrees , 3 degrees ), and 2 degrees HCG fate specification is mediated by LIN-12/Notch. We show that 2 degrees HCG specification depends on the
presence of a cell with the 1 degrees fate. We also provide evidence that Wnt signaling via the Frizzled-like Wnt receptor LIN-17 act to specify the 1 degrees and 2 degrees HCG fate. A requirement for EGF signaling during 1 degrees fate specification is seen only when LIN-17 activity is compromised. In addition, activation of the EGF pathway decreases dependence on LIN-17 and causes ectopic hook development. Our results suggest that WNT plays a more significant role than EGF signaling in specifying HCG fates, whereas in VPC specification EGF signaling is the major inductive signal. Nonetheless, the overall logic is similar in the VPCs and the HCG: EGF and/or WNT induce a 1 degrees lineage, and LIN-12/NOTCH induces a 2 degrees lineage. Wnt signaling is also required for execution of the 1
degrees and 2 degrees HCG lineages. lin-17 and bar-1/beta-catenin are preferentially expressed in the presumptive 1 degrees cell P11.p. The dynamic subcellular localization of BAR-1-GFP in P11.p is concordant with the timing of HCG fate determination.
2: Cancer Sci 2009
Jan;
Epidermal growth factor receptor lacking C-terminal autophosphorylation sites retains signal transduction and high sensitivity to epidermal growth factor receptor tyrosine kinase inhibitor.
[Abstract] Constitutively active mutations of epidermal growth factor receptor (EGFR) (delE746_A750) activate downstream signals, such as ERK and Akt, through the phosphorylation of tyrosine residues in the C-terminal region of EGFR. These pathways are thought to be important for cellular sensitivity to EGFR tyrosine kinase inhibitors (TKI). To examine the correlation between phosphorylation of the tyrosine residues in the C-terminal region of EGFR and cellular sensitivity to EGFR TKI, we used wild-type (wt) EGFR, as well as the following constructs: delE746_A750 EGFR; delE746_A750 EGFR with substitution of seven tyrosine residues to phenylalanine in the C-terminal region; and delE746_A750 EGFR with a C-terminal truncation at amino acid 980. These constructs were
transfected stably into HEK293 cells and designated HEK293/Wt, HEK293/D, HEK293/D7F, and HEK293/D-Tr, respectively. The HEK293/D cells were found to be 100-fold more sensitive to EGFR TKI (AG1478) than HEK293/Wt. Surprisingly, the HEK293/D7F and HEK293/D-Tr cells, transfected with EGFR lacking the C-terminal autophosphorylation sites, retained high sensitivity to EGFR TKI. In these three high-sensitivity cells, the ERK pathway was activated without ligand stimulation, which was inhibited by EGFR TKI. In addition, although EGFR in the HEK293/D7F and HEK293/D-Tr cells lacked significant tyrosine residues for EGFR signal transduction, phosphorylation of Src homology and collagen homology (Shc) was spontaneously activated in these cells. Our results indicate that tyrosine residues in the
C-terminal region of EGFR are not required for cellular sensitivity to EGFR TKI, and that an as-yet-unknown signaling pathway of EGFR may exist that is independent of the C-terminal region of EGFR. (Cancer Sci 2009).
3: Bioconjug Chem 2009
Jan;Vol 20(1)
Surface-anchoring of spontaneously dimerized epidermal growth factor for highly selective expansion of neural stem cells.
[Abstract] To develop culture substrates for use in selective expansion of neural stem cells (NSCs), epidermal growth factor (EGF)-containing chimeric proteins were designed and synthesized by means of recombinant DNA technology. The chimeric proteins consisted of three components including an EGF domain, an alpha-helical oligopeptide, and a hexahistidine sequence. Two different alpha-helical oligopeptides were separately incorporated into chimeric proteins. Structural analyses by native gel electrophoresis and circular dichroism spectroscopy revealed that the heterodimer of these proteins was spontaneously formed through coiled-coil association of the alpha-helical oligopeptides. The monomeric and dimeric forms of these chimeric proteins were immobilized to the
glass-based substrate via coordinate bonding between the hexahistidine and Ni(II) ions fixed on a substrate. The results of cell culture assays with NSCs showed that cells proliferated most rapidly and selectively on a substrate with the surface-anchored EGF dimer. The rate of cell proliferation on the surface with dimeric EGF was 1.3-2.0 times higher on the surfaces with monomeric EGF. In addition, the content of stem cells, determined 96 h after cell seeding, was highest on the surface with dimeric EGF (98%) among the surfaces studied (90-97% on surfaces with monomeric EGF). The observed growth rate and the stem cell content on the surface with EGF dimer were far beyond those in the standard neurosphere culture. The effect of surface-anchored dimeric EGF may be attributed to the enhanced
dimerization of EGF-EGF receptor complexes leading to efficient signaling for mitogenic activity. We conclude that surface-anchoring of the EGF dimer provides an excellent substrate that allows the highly efficient expansion of NSCs.
4: Endocr Relat Cancer 2009
Jan;
Obestatin stimulates Akt signalling in gastric cancer cells through {beta}-arrestin-mediated epidermal growth factor receptor transactivation.
[Abstract] Obestatin was identified as a gut peptide encoded by the ghrelin gene that interacts with the G protein-coupled receptor, GPR39. In this work, a sequential analysis of its transmembrane signalling pathway has been undertaken to characterize the intracellular mechanisms responsible for Akt activation. The results show that Akt activation requires the phosphorylation of T308 in the A-loop by the phosphoinositide-dependent kinase 1 (PDK1) and S473 within the HM by the mammalian target of rapamycin (mTOR) kinase complex 2 (mTORC2: Rictor, mLST8, mSin1, mTOR kinase) with participation neither of Gi/o-protein nor Gbetagamma dimers. Obestatin induces the association of GPR39/beta-arrestin 1/Src signalling complex resulting in the transactivation of the epidermal
growth factor receptor (EGFR) and downstream Akt signalling. Upon administration of obestatin, phosphorylation of mTOR (S2448) and p70S6K1 (T389) rise with a time course that parallels that of Akt activation. Based on the experimental data obtained, a signalling pathway involving a beta-arrestin 1 scaffolding complex and EGFR to activate Akt signalling is proposed.
Domain Info
GeneBank Entry:
NM_001963
Protein Accession No.:
NP_001954
Protein Sequence:
NSDSE CPLSH DGYCL HDGVC MYIEA LDKYA CNCVV GYIGE RCQYR DLKWW ELR
Transcript Info
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