Construction of Synthetic Intact Human Parathyroid Hormone Gene and Testing the Transformation Efficiency and Expression of it in E. coli strains

  • Adnan Al-Badran University of Basra, College of Science, Department of Biology, Basra, Iraq
  • Rafeef A. Abdul-Jabbar University of Basra, College of Pharmacy, Department of Clinical Laboratory Science, Basra, Iraq
Keywords: synthetic gene, synthetic human parathyroid hormone gene, SOE PCR.


 human parathyroid hormone gene (hPTH gene) is responsible for preproparathyroid hormone production (biologically inactive hormone) which further modified to form the biologically active hormone, a synthetic hPTH gene with 289bp which cod for active intact hPTH was constructed by Five long primers by splicing overlap extension PCR (SOE), the gene contain a de generated codon, and have two recognition site of restriction enzymes in both ends of the Gene, and a stop codon. The synthetic hPTH gene was transformed in two E. coli strains DH5α and BL21 (DE3) with transformation efficiency6.6* 106 and 3.4* 106 for both isolatesrespectively. And the transformation of gene was detected by extraction of constructed plasmid and amplification of cloning gene on it, the expression of the cloning gene in the BL21 (DE3) was detected by performing Real Time PCR which gave a Ct value of about (23.46). That makes the synthetic gene suitable for direct expression of human active protein inside the bacterial cells.


[1] Bilezikian, J. P, Marcus, R., Levine, M. A., Marcocci, C, Silverberg, S.J, Potts, J. T. The Parathyroids: Basic and Clinical Concepts. Academic Press.3rd edition, Print Book ISBN: 9780123971661. EBook ISBN: 9780123977908, 2014. Pages: 946.
[2] Goswami, R., Mohapatra, T., Gupta, N., Rani, R., Tomar, N., Dikshit, A., & Sharma, R. K. Parathyroid hormone gene polymorphism and sporadic idiopathic hypoparathyroidism. Journal of Clinical Endocrinology and Metabolism, 89(10), 4840–4845, 2004.
[3] Khow, O., & Suntrarachun, S. Strategies for production of active eukaryotic proteins in bacterial expression system. Asian Pacific Journal of Tropical Biomedicine, 2(2), 159–162. 2012.
[4] Marsic, D., Hughes, R. C., Byrne-Steele, M. L., & Ng, J. D. PCR-based gene synthesis to produce recombinant proteins for crystallization. BMC Biotechnology, 8, 44. 2008.
[5] Li, L., Wu, L. P., & Chandrasegaran, S. Functional domains in Fok I restriction endonuclease. Proceedings of the National Academy of Sciences of the United States of America, 1992. 89(10), 4275–9.
[6] Stemmer, W.P., Crameri, A., Ha, K.D., Brennan, T. M. and Heyneker, H. L. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxy ribonucleotides. Gene. 1995, 16; 164(1):49-53.
[7] Gao, X., Yo, P., Keith, A., Ragan, T. J., & Harris, T. K. Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for high-fidelity assembly of longer gene sequences. Nucleic Acids Research, 31(22), 143-146. 2003.
[8] Rouillard, J. M., Lee, W., Truan, G., Gao, X., Zhou, X., & Gulari, E. Gene2Oligo: Oligonucleotide design for in vitro gene synthesis. Nucleic Acids Research, 32(WEB SERVER ISS.), 176–180. 2004.
[9] Xiong, A. S., Yao, Q. H., Peng, R. H., Li, X., Fan, H. Q., Cheng, Z. M., & Li, Y. A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences. Nucleic Acids Research, 32(12): 98-104. 2004.
[10] Horton, R. M., Cai, Z., Ho, S. N., and Pease L. R. Gene Splicing by Overlap Extension: Tailor-Made Genes Using the Polymerase Chain Reaction. Reprinted from BioTechniques. 8(5):528-535. 1990.
[11] Heckman, K. L., & Pease, L. R. Gene splicing and mutagenesis by PCR-driven overlap extension. Natural Protocol, 2(4), 924–932. 2007.
[12] Liu, Q., Lin, J., Liu, M., Tao, X., Wei, D., Ma, X., & Yang, S. Large scale preparation of recombinant human parathyroid hormone 1-84 from Escherichia coli. Protein Expression and Purification, 54(2), 212–219. 2007.
[13] Salih A. A., Mehdi K. H., A.-B. A. I. Cloning of Human Insulin Gene in Bacterial Expression System. LAP LAMBERT Academic Publishing, 2013.
[14] Kronenberg, H. M., Samuel, R. Nussbaum, T. D. Recombinant DNA method for production of parathyroid hormone. 1990. WO 1990014415 A1. Patent WO1990014415A1.
[15] Wei, X., Kuhn, D. N., & Narasimhan, G. Degenerate primer design via clustering.. Proceedings of the 2003 IEEE Bioinformatics Conference, CSB,2003, 75–83.
[16] Angov, E. Codon usage: Nature’s roadmap to expression and folding of proteins. Biotechnology Journal, 6(6), 650–659. 2011.
[17] Naimov S., Dukiandjiev S. and de Maagd R. A. A rapid pcr based method for gene synthesistrol. Proceedings Of The Balkan Scientific Conference Of Biology In Plovdiv (Bulgaria) From, 2005(May), 277–282.
[18] Nguyen D.H. The Advantages of Using Sticky End Enzymes. 2016.
[19] Griffiths AJF, Miller JH, Suzuki DT, et. An Introduction to Genetic Analysis. 7th edition. W. H. Freeman and Company. 2000.
[20] Novagen. competent cells. User Protocol TB009 Rev. F 0104 © EMD Biosciences, Inc.,1-23, 2004.
[21] Felthauser, A. Preparation of DH5a competent cells. Solutions, 2002.10–12.
[22] Rosano, G. L., & Eduardo, C. A. Recombinant protein expression in Escherichia coli: Advances and challenges. Frontiers in Microbiology, 5(APR), 1–17. 2014.
[23] Sigma Aldrich. TRI reagent protocol. User Manual - Sigma Aldrich, (10814), 1–2, 2014.
[24] White, P. Evaluating Concentration and Purity of RNA. Functional GENOMIC CORE, 2004. 6–7.
[25] QIAGEN. October For fast and highly sensitive one-step RT-PCR Sample & Assay Technologies QIAGEN Sample and Assay Technologies, 2012 (October).
[26] Bustin, S. a. Real-Time Reverse Transcription PCR. Encyclopedia of Diagnostic Genomics and Proteomics, 2005. 718–726.
[27] Pfaffl, M. Livestock Transcriptomics: Quantitative mRNA Analytics in Molecular Endocrinology and Physiology. Accessed at February, 2006. (January). Retrieved from Retrieved from biology/tri-reagent.html.
[28] Downey, N. Interpreting melt curves: An indicator, not a diagnosis Researchers. 2014. Retrieved from
[29] Vermasvuori, R.. Production of recombinant proteins and monoclonal antibodies - Techno-economical evaluation of the production methods. English, 2009. 1–117.