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Molecular Biology Reports

, Volume 41, Issue 3, pp 1445–1451 | Cite as

Expression line approach to recombinant human epidermal growth factor into the yeast, Pichia pastoris from Huh-7 cell line

  • Mohsin Ahmad Khan
  • Faidad Khan
  • Nadeem Ahmad
  • Muhammad Islam Khan
  • Ahmad Usman Zafar
  • Tayyab Husnain
Article

Abstract

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.

Keywords

Human epidermal growth factor Human hepatoma cell line Pichia pastoris Polymerase chain reaction 

Notes

Acknowledgments

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.

References

  1. 1.
    Cohen S, Carpenter G (1975) Human epidermal growth factor: isolation and chemical and biological properties. PNAS 72:1317–1321PubMedCrossRefGoogle Scholar
  2. 2.
    Fraser JK, Zhu M, Wulur I, Alfonso Z (2008) Adipose-derived stem cells. Methods Mol Biol 449:59–67PubMedGoogle Scholar
  3. 3.
    Mark JK, Mourad M, Ole M, Walter P, Yong YW, Ronald EB, Peter JH, Robert MH, Jorg H (2006) An unusual mode of concerted evolution of the EGF-TM7 receptor chimera EMR2. FASEB J 20(14):2582CrossRefGoogle Scholar
  4. 4.
    Engler DA, Hauser MR, Cook JS, Niyogi SK (1991) Aromaticity at position 37 in human epidermal growth factor is not obligatory for activity. Mol Cell Biol 11(5):2425PubMedCentralPubMedGoogle Scholar
  5. 5.
    Graeme IB, Noel MF, Michelle MS, Mary AW, Daniel C, Lailing K, Mickey SU, Leslie BR, Ray SP (1986) Human epidermal growth factor precursor: cDNA sequence, expression in vitro and gene organization. Nucleic Acids Res 14(21):8427–8446CrossRefGoogle Scholar
  6. 6.
    Carpenter G, Cohen S (1979) Epidermal growth factor. Annu Rev Biochem 48:193–216PubMedCrossRefGoogle Scholar
  7. 7.
    Anna MW (2005) Prospects for developmental biology and cell therapy. Physiol Rev 85:635–678CrossRefGoogle Scholar
  8. 8.
    Strutz F, Michael Z, Fuad NZ, Chang-Qing Y, Raghu K, Gerhard AM, Eric GN (2002) Role of basic fibroblast growth factor-2 in epithelial mesenchymal transformation. Kidney Int 61(5):1714–1728PubMedCrossRefGoogle Scholar
  9. 9.
    Schuldiner MO, Joseph IE, Douglas AM, Nissim B (2000) Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc Natl Acad Sci 97(21):11307–11312PubMedCrossRefGoogle Scholar
  10. 10.
    Robert MC, Anthony JW, Martin B, Annalisa P, Michael JT, Lain DC, Harry G, Brian S (1987) The solution structure of human epidermal growth factor. Nature 327:339–341CrossRefGoogle Scholar
  11. 11.
    Umeoka K, Naoko S, Kenichi O, Tahara S, Reiko K, Shoichiro I, Manabu N, Takeshi W, Yoshiyuki OAT (2001) Immuno histochemical analysis of RCAS1 in human pituitary adenomas. Mod Pathol 14(12):1232–1236PubMedCrossRefGoogle Scholar
  12. 12.
    Adams JC (2001) Review cell-matrix contact structures. Cell Mol Life Sci 58(3):371–392PubMedCrossRefGoogle Scholar
  13. 13.
    Chung KF (2001) Cytokines in chronic obstructive pulmonary disease. Eur Respir J 18(34):50–59CrossRefGoogle Scholar
  14. 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. 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. 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
  17. 17.
    John S, Cook E, Ian F, Sally P, Roger D, Michael AW, Mike D, David MJ, John FP, Thakor P, Hilary L, Leslie DB (1982) Chemical synthesis and cloning of a gene for human urogastrone. Nucleic Acids Res 10(15):4467–4482CrossRefGoogle Scholar
  18. 18.
    Patil MA, Zhang J, Ho C, Cheung ST, Fan ST, Chen XP (2006) Hedgehog signaling in human hepatocellular carcinoma. Cancer Biol Ther 5(1):111–117PubMedCrossRefGoogle Scholar
  19. 19.
    Brett DL, Matthew JE, Andrew JS, Benno W, Timothy LT, Christopher CL, Toshiaki M, Richard OH, Dennis RB, Jane AM, Charles MR (2005) Complete replication of hepatitis c virus in cell culture. Science 309(5734):623–626CrossRefGoogle Scholar
  20. 20.
    Takimoto R, Kato J, Terui T, Takada K, Kuroiwa G, Wu J, Ohnuma H, Takahari D, Kobune M, Sato Y, Takayama T, Matsunaga T, Niitsu Y (2005) Augmentation of antitumor effects of p53 gene therapy. Cancer Biol Ther 4(4):421–428PubMedCrossRefGoogle Scholar
  21. 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. 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. 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
  24. 24.
    Chomczynski P, Mackey K (1995) Modification of the trizol reagent procedure for isolation of RNA from polysaccharide and proteoglycan-rich sources. Biotechniques 19(6):942–945PubMedGoogle Scholar
  25. 25.
    Steve R, Helen JS (2000) Primer 3, http://frodo.wi.mit.edu/primer3/ Bioinformatics methods and protocols methods in molecular biology, 365–386
  26. 26.
    Benson DA, Karsch-Mizrachi I, Clark K, Lipman DJ, Ostell J, Sayers EW (2012) GenBank. Nucleic Acids Res 40:D48–D53PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Sambrook J, Russell DW (2006) Preparation and transformation of competent E. coli using calcium chloride. Cold Spring Harb Protoc. doi: 10.1101/3932 Google Scholar
  28. 28.
    Baron M, Reynes JP, Stassi D, Tiraby G (1992) A selectable bifunctional b galactosidase: phleomycin-resistance fusion protein as a potential marker for eukaryotic cells. Gene 114:239–243PubMedCrossRefGoogle Scholar
  29. 29.
    Plyler TR, Vallejos CE (2000) Method for cloning restriction fragments containing the termini of BAC Inserts. Biotechniques 28(5):1012–1016PubMedGoogle Scholar
  30. 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. 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. 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
  33. 33.
    Takanori O, Shunji S, Ken-Ichi M, Toru F, Koji Y, Makari Y, Gakuzo T, Tetsuo M (1985) Synthesis and secretion of human epidermal growth factor by Escherichia coli. Proc NatI Acad Sci 82(21):7212–7216CrossRefGoogle Scholar
  34. 34.
    John MC, Graham HC, Mickhael JP, Mark ER (1991) Secretion of human epidermal growth factor from S. cerevisiae using synthetic leader sequences. Gene 106(2):267–271CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Mohsin Ahmad Khan
    • 1
  • Faidad Khan
    • 1
  • Nadeem Ahmad
    • 1
  • Muhammad Islam Khan
    • 1
  • Ahmad Usman Zafar
    • 1
  • Tayyab Husnain
    • 1
  1. 1.National Centre of Excellence in Molecular BiologyUniversity of the PunjabLahorePakistan

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