African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12191

Full Length Research Paper

Recombinant expression and purification of L2 domain of human epidermal growth factor receptor

Elnaz Salehi1, Safar Farajnia2,3*, Kazem Parivar1, Behzad Baradaran4, Jafar Majidi 4, Yadollah Omidi5 and Nazli Saeedi3
1Science and Research Branch, Islamic Azad University of Tehran, Tehran, Iran. 2Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 3Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 4Immunology Department, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. 5Pharmaceutical Nanotechnology Research Center, Tabriz University of Medical sciences, Tabriz, Iran.
Email: [email protected]

  •  Accepted: 07 May 2010
  •  Published: 31 August 2010

Abstract

Epidermal growth factor receptor (EGFR) is one of the key molecules in cell growth and multiplication and plays an important role in some malignant processes. L2 domain of extra-cellular part of this receptor involved in ligand binding and its inhibition can prevent activation of related signaling pathways. The aim of the present study was cloning and expressing the fragment coding for L2 region of human EGFR for the production of recombinant L2 protein. The total RNA from A431 cells line was extracted and used for amplification of the sequence coding for L2 domain of EGFR by reverse transcriptase-polymerase chain reaction (RT-PCR) technique. The product was cloned into the PGEM-T vector and used for sequencing. In the next step, the insert was removed from the PGEM-T vector and subcloned into the PET22 expression vector. The expression construct was transformed into the Escherichia coli BL21 (DE3) and recombinant protein expression was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Analyzing the expression of produced recombinant protein showed that the recombinant L2 can be highly expressed by this expression system. This recombinant protein can be used for the production of specific mAb, screening for specific ligands and competitive inhibition of the EGFR.

 

Key words: Human epidermal growth factor receptor, domain L2, recombinant expression.

Abbreviation

EGFR, Epidermal growth factor receptor; RT-PCR, reverse transcriptase-polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresisCR, cysteine rich; DEPC, diethylpyrocarbonate;BLAST, basic alignment search toolsLB, Luria-BertaniIPTG, isopropyl-D-thiogalactopyranoside.