Bispecific antibodies have been exploited as both cancer immunodiagnostics and cancer therapeutics, which have shown promises in clinical trials in cancer imaging and therapy. To improve the anti-tumor effect, an scDb (bispecific single-chain diabody) was constructed from the variable domain genes of two scFvs (single-chain variable fragment antibodies) directed against human EGFR (epidermal growth factor receptor) and VEGFR2 (vascular endothelial growth factor receptor 2) extracellular domains. The anti-EGFR/ anti-KDR scDb was constructed into pHEN2 plasmid and expressed in Escherichia coli HB2151 host. After purification by one-step affinity chromatography of IMAC, scDb protein was characterized by Western blotting. The yield of scDb protein was 570 microg per liter medium. scDb bound to EGFR as efficiently as the parental antibody scFv-E10, while a little bit weaker than the parental antibody scFv-AK404R when bound to KDR. In conclusion, the scDb protein could bind both EGFR and KDR specifically and could be applied for further anti-tumor research.
Antibodies, Bispecific ; biosynthesis ; genetics ; Escherichia coli ; metabolism ; Humans ; Plasmids ; Protein Binding ; Receptor, Epidermal Growth Factor ; immunology ; Single-Chain Antibodies ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; immunology

The epithelial growth factor receptor interference (EGFRi) was obtained by synthetic primers. Overlapping PCR was used to produce EGFRi-IL-24 fusion gene, which is linked by Gly4Ser3. After sequence analysis, EGFRi-IL-24 was cloned into expression vector pPIC9k; EGFRi-IL-24/pPIC9k was linearized with SacI,and then transformed to electroporated pastoris GS115. Subsequently, positive clone was selected by G418 and PCR, and its phenotype was determined by SDS-PAGE and MTT assay. The results demonstrated that EGFRi-IL-24 protein was expressed and shown to have the potential for use in researches of its biological function and in clinical application.
Antineoplastic Agents ; pharmacology ; Genetic Vectors ; genetics ; Humans ; Interleukins ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

A DNA fragment encoding extracellular domain of mouse epidermal growth factor receptor (EGFR) was obtained by PCR from a previous recombinant plasmid. The DNA fragment was then ligated into prokaryotic expression vector, and expressed in Escherichia Coli. The recombinant protein was purified under denature conditions by affinity chromatography, and refolded with gradient dialysis. The recombinant protein could produce antibodies to recognize extracellular domain and full-length of mouse EGFR, and form homodimer in the presence of EGF detected by western blot analysis. These findings provide evidence that the renatured recombinant extracellular domain of mouse epidermal growth factor receptor is immunogenetic and may be important for further application of this protein in functional and immunological research.
Animals ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Mice ; Plasmids ; Receptor, Epidermal Growth Factor ; biosynthesis ; chemistry ; genetics ; isolation & purification ; Transfection

The human epidermal growth factor receptor (EGFR) extracellular region (residues 1-621) consists of four subdomains, i.e. L1, S1, L2, and S2. The L2 domain (EGFR-L2) is composed of residues 311-479 and plays a major role in ligand-binding. Due to the high content of cysteine residues (42 cysteines) in the S1 and S2 domains, it is quite difficulty to get a correctly refolded product of the complete EGFR extracellular domain. In contrast, only 4 cysteine residues are present in EGFR-L2 domain. The aim of the present study is to prepare a soluble EGFR-L2 domain from the recombinant protein inclusion body overexpressed in Escherichia coli (E. coli). DNA fragment encoding EGFR-L2 containing a polyhistidine-tag at the carboxyl terminus was amplified by PCR from the cDNA of EGFR extracellular region, and was inserted into pET-3c to construct the prokaryotic expression vector. The target protein was highly expressed in E. coli BL21 (DE3) strain and was only present in the inclusion body as revealed by immunoblotting analysis. No soluble product could be refolded through dilution or stepwise dialysis strategies. However, on-column refolding of denatured EGFR-L2 bound to Ni2+ -NTA produced a soluble one. Furthermore,the soluble EGFR-L2 was simultaneously purified to high purity (>95%) through eluting from the same Ni2+ -NTA column with a linear imidazole gradient. The refolded EGFR-L2 had specific binding activity with the cognate ligand EGF, although its affinity was low. These results suggest that a polyhistidine-tag fused with a recombinant protein facilitate not only the purification but also the renaturation of the target product through on-column refolding. Besides, this refolding strategy may be suitable for the preparation of those recombinant proteins which are hard to refold through conventional approaches.
Escherichia coli ; genetics ; metabolism ; Humans ; Inclusion Bodies ; genetics ; metabolism ; Protein Folding ; Receptor, Epidermal Growth Factor ; biosynthesis ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

This experiment was designed to study the effect of 131I-recombinant human epidermal growth factor (131I-rhEGF) on the growth of tumor in nude mice loaded with human breast cancer. Bioactivity of 131I-rhEGF and uptake of 131I-rhEGF in breast cancer tissue were verified using biodistribution experiment of 131I-rhEGF in the nude mice loaded with human breast cancer. The effect of 131I-rhEGF on the growth of tumor was assessed via the growth experiment of tumor in the nude mice loaded with human breast cancer. The ultrastructural change of the tumor cell treated with 131I-rhEGF was observed under transmission electron microscope, and the pathological change of the tumor tissue treated with 131I-rhEGF was detected by biopsy. The results showed that the tumor tissue of nude mice bearing human breast cancer obviously takes in 131I-rhEGF; that intravenous administration and intratumoral administration of 131I-rhEGF both obviously inhibit the growth of tumor, the inhibition rates (82.00% and 80.70%) being remarkably higher than that of 131I (7.49%) and that of 131I-HSA (6.91%) (P<0.05); and that intravenous and intratumoral administration of 131I-rhEGF both obviously damage and kill tumor cells. Therefore, 131I-rhEGF can inhibit the growth of human breast cancer cell in nude mice; it is a potential receptor-mediated radioactivity targeting drug for treating breast cancer.
Animals ; Breast Neoplasms ; pathology ; ultrastructure ; Drug Delivery Systems ; Epidermal Growth Factor ; biosynthesis ; genetics ; pharmacology ; Female ; Humans ; Iodine Radioisotopes ; administration & dosage ; pharmacology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Receptor, Epidermal Growth Factor ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology

Epidermal growth factor receptor (EGFR) and its ligands (EGF and TGFalpha) are over-expressed in a variety of tumors. Immunization EGF-carrier protein inhibits tumor growth through abrogating binding of EGF to EGFR. Here, a chimeric protein of EGF and TGFalpha (E5T) was genetically fused to Staphylococcal enterotoxin A (SEA), a bacterial superantigenic protein which promotes humoral B cell response through enhancement of Ag-specific CD4 T cells activity. The resulted fusion proteins were expressed in Escherichia coli and purified though metal chelating affinity chromatography. Immunization of E5T-mSEA fusion protein in mice induced production of high titers antibodies, which recognize both EGF and TGFalpha. Anti- E5T-mSEA serum at dilution of 1:10 significantly inhibited growth of A431 cell lines but had little effect on 293T cell lines.
Amino Acid Sequence ; Animals ; Cancer Vaccines ; biosynthesis ; immunology ; Cell Line, Tumor ; Enterotoxins ; biosynthesis ; genetics ; Epidermal Growth Factor ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunization ; Mice ; Mice, Inbred C57BL ; Molecular Sequence Data ; Random Allocation ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Transforming Growth Factor alpha ; biosynthesis ; genetics

OBJECTIVETo observe the growth and cell cycle changes of nasopharyngeal carcinoma cell 5-8F in response to silencing of the expression of epidermal growth factor receptor (EGFR) with RNA interference (RNAi), and explore the possible relationships between EGFR and the occurrence, differentiation and progression of nasopharyngeal carcinoma.

METHODThree EGFR-specific small interfering RNAs (siRNAs) were obtained by in vitro transcription and synthesis and were transiently transfected into 5-8F cells. The EGFR expression levels in the transfected cells were detected by reverse transcription (RT)-PCR and Western blotting, respectively. After EGFR expression silencing, the growth and cell cycle changes of the cells were observed.

RESULTSEGFR mRNA contents and protein levels decreased by approximately 67.5% and 77%, respectively, after RNAi of 5-8F cells, and the cell proliferation decreased and cell cycle arrest at G1 phase occurred in association with EGFR silencing.

CONCLUSIONEGFR silencing by RNAi can reduce the proliferation of nasopharyngeal carcinoma cells and induce cell cycle arrest at G1 phase, which sheds light on the possible use of RNAi for further investigation of the pathogenesis and gene therapy of nasopharyngeal carcinoma.

Cell Line, Tumor ; Cell Proliferation ; Genetic Therapy ; Humans ; Nasopharyngeal Neoplasms ; genetics ; metabolism ; pathology ; RNA Interference ; RNA, Small Interfering ; genetics ; Receptor, Epidermal Growth Factor ; biosynthesis ; genetics ; Transfection

Animals ; Carcinoma, Non-Small-Cell Lung ; genetics ; metabolism ; Humans ; Lung Neoplasms ; genetics ; metabolism ; Molecular Imaging ; Mutation ; Positron-Emission Tomography ; RNA, Messenger ; metabolism ; Receptor, Epidermal Growth Factor ; biosynthesis ; genetics

OBJECTIVETo create a gene transfer vehicle for targeting gene therapy of cancer with epidermal growth factor receptor overexpressing.

METHODSEncoding sequences of the first domain of histone gene (H1) and EGF C-loop (EGFc) were obtained by PCR amplification. These two DNA fragments were ligated by EcoR I site, and cloned and sequenced. E. coli expression vector of the fusion gene was then constructed. The fusion protein H1EGFc was purified by specific band isolation from SDS-PAGE.

RESULTSThe molecular weight of purified protein was consistent with the designed request. Its purity reached 94.02%.

CONCLUSIONA fusion protein H1EGFc was expressed and purified.

Amino Acid Sequence ; Base Sequence ; Escherichia coli ; genetics ; Gene Transfer Techniques ; Genetic Therapy ; Genetic Vectors ; Histones ; biosynthesis ; genetics ; Humans ; Molecular Sequence Data ; Protein Engineering ; Receptor, Epidermal Growth Factor ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

OBJECTIVETo explore the effects of antisense epidermal growth factor receptor (EGF-R) oligodeoxynucleotides on ultraviolet-induced c-jun activity of keratinocytes after EGF-R oligodeoxynucleotides transfect to HaCaT in vitro.

METHODSc-jun DNA binding activity after ultraviolet-B (UVB) irradiation and EGF-R oligodeoxynucleotides transfection were determined with a highly sensitive and specific colorimetric method. After EGF-R oligodeoxynucleotides transfection, the mRNA level of EGF-R was detected by reverse transcription polymerase chain reaction method.

RESULTSCompared with control groups, c-jun activity increased significantly in UVB (10, 20, 30 mJ/cm2) irradiation groups (P < 0.05). EGF-R mRNA and c-jun activities induced by UVB were inhibited after the keratinocytes were transfected with EGF-R antisense oligodeoxynucleotides at 2, 4 and 8 microg/ml concentrations (P < 0.01).

CONCLUSIONThe ultraviolet-induced c-jun activity of keratinocytes can be mediated by EGF-R and inhibited by EGF-R antisense oligodeoxynucleotides, which is transfected to keratinocytes and mediated by lipofectamine.

Cell Line ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Keratinocytes ; drug effects ; metabolism ; radiation effects ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Receptor, Epidermal Growth Factor ; biosynthesis ; genetics ; Transfection ; Ultraviolet Rays