Expression line approach to recombinant human epidermal growth factor into the yeast, Pichia pastoris from Huh-7 cell line
- 321 Downloads
Beta-urogastrone also known as human epidermal growth factor is a key member of epidermal growth factor family having role in cell proliferation and differentiation in vivo as well as in vitro. Human epidermal growth factor gene has been isolated from different tissues but the method of isolation is technically difficult and complicated as it deals with biopsies. Here we isolated mature partial human epidermal growth factor gene from Huh-7 cell line, amplified and abridged toward mature coding region with three steps PCR, sequenced for homology with wild type human epidermal growth factor gene, inbuilt with sites of interest and cloned in Pichia pastoris for expression study. Isolated mature human epidermal growth factor gene from Huh-7 cell line showed 100 % sequence homology to wild type human epidermal growth factor gene and gives the native expression for human epidermal growth factor peptide. In this study we report that Huh-7 cell line is an easy source for the particular gene of human epidermal growth factor isolation and we are also suggesting P. pastoris is an expression system to produce recombinant human epidermal growth factor of the therapeutic importance resembling to the natural human system.
KeywordsHuman epidermal growth factor Human hepatoma cell line Pichia pastoris Polymerase chain reaction
We thank to Molecular Medicine Lab (CEMB) for providing Huh-7 cell line. Thanks to Ahmad Usman Zafar, Nadeem Ahmad, Muhammad Islam Khan and Tayyab Hussnain for thoughtful discussions and critical reading of the manuscript.
Conflict of interest
The authors declare that they have no competing interests.
- 14.Philip JA, Michael BC, Cherie MC, Kenneth H (2009) Method of using cytokine assay to diagnose, treat and evaluate inflammatory and autoimmune diseases. Google PatentsGoogle Scholar
- 15.Mieko K, Shigeaki T (1989) Topographic analysis of human epidermal growth factor by monospecific antibodies and synthetic peptides. Oxford Journals. Life Sciences. J Biochem 106(1):87–89Google Scholar
- 16.Beat MS, Anna F, Eric F, Albert J (1994) Liver expression of epidermal growth factor RNA. J Biol Chem 269(31):9667–9670Google Scholar
- 21.Jie L, Lilianna S, Ruben MB, Jan N, Liping H, Natascha N, Steven D, Liegang L, Ulrich S, Andreas KN (2012) Comparative analysis of phase I and II enzyme activities in 5 hepatic cell lines identifies Huh-7 and HCC-T cells with the highest potential to study drug metabolism. Arch Toxicol 86(1):87–95CrossRefGoogle Scholar
- 22.Bao-Zhu Y, Mark JM, Catherine LK, Drazen BZ, Snorri ST, Nicholas CP (1998) Cloning, characterization and chromosomal localization of a gene frequently deleted in human liver cancer (DLC-1) homologous to Rat RhoGAP. Cancer Res 58:2196Google Scholar
- 23.Tanabe KK, Lemoine A, Finkelstein DM, Kawasaki H, Fujii T, Chung RT, Lauwers GY, Kulu Y, Muzikansky A, Kuruppu D, Lanuti M, Goodwin JM, Azoulay D, Fuchs BC (2008) Epidermal growth factor gene functional polymorphism and the risk of hepatocellular carcinoma in patients with cirrhosis. JAMA 299(1):53–60PubMedCrossRefGoogle Scholar
- 25.Steve R, Helen JS (2000) Primer 3, http://frodo.wi.mit.edu/primer3/ Bioinformatics methods and protocols methods in molecular biology, 365–386
- 30.Kerri AK, Helen SH, Douglas RD, Constance LC, Budowle B (2002) An improved method for post-PCR purification for mtDNA sequence analysis. J Forensic Sci 47(4):811–818Google Scholar
- 31.Watanabe K, Emoto N, Sunohara M, Kawakami M, Kage H, Nagase T, Ohishi N, Takai D (2010) Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfate restriction analysis. Biochem Biophys Res Commun 399(3):422–424PubMedCrossRefGoogle Scholar
- 32.Frederick MA, Roger B, Robert EK, David DM, Seidman JG, John AS, Kevin S (1999) Short protocols in molecular biology, 4th edn. Wiley, New YorkGoogle Scholar