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Expression of Biologically Active Crustacean Hyperglycemic Hormone (CHH) of Penaeus monodon in Pichia pastoris

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Abstract

Crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) are members of a major peptide family produced from the X-organ sinus gland complex in the eyestalk of crustaceans. This peptide family plays important roles in controlling several physiologic processes such as regulation of growth and reproduction. In this study the complementary DNA encoding a peptide related to the CHH/MIH/GIH family (so-called Pem-CMG) of the black tiger prawn Penaeus monodon was successfully expressed in the yeast Pichia pastoris under the control of the AOX1 promoter. The recombinant Pem-CMG was secreted into the culture medium using the α-factor signal sequence; of Saccharomyces cerevisiae without the Glu-Ala-Glu-Ala spacer peptide. The amino terminus of the recombinant Pem-CMG was correctly processed as evidenced by amino-terminal peptide sequencing. The recombinant Pem-CMG was purified by reverse-phase high-performance liquid chromotography and used in a biological assay for CHH activity. The final yield of the recombinant Pem-CMG after purification was 260 µg/L of the culture medium. Both crude and purified recombinant Pem-CMG produced from P. pastoris showed the ability to elevate the glucose level in the hemolymph of eyestalk-ablated P. monodon, which demonstrates that Pem-CMG peptide functions as hyperglycemic hormone in P. monodon.

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Acknowledgements

We thank Dr. Duncan Smith for his critical reading of this manuscript and Dr. Paisarn Sithigorngul, Department of Biology, Faculty of Science, Srinakharinwirot University, for his suggestion on protein purification and the biological assay. This work was financially supported by Thailand Research Fund (TRF) and Thailand National Center for Biotechnology and Genetic Engineering, National Science and Technology Development Agency (NSTDA). S.T. was funded by Graduated Fellowship Program, NSTDA, 1999–2001.

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Authors and Affiliations

  1. Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakhon Pathom 73170, Thailand

    Supattra Treerattrakool, Apinunt Udomkit, Burachai Sonthayanon & Sakol Panyim

  2. BIOTEC Training Center of Genetic Engineering and Biotechnology at Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakhon Pathom 73170, Thailand

    Lily Eurwilaichitr

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  1. Supattra Treerattrakool
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  2. Apinunt Udomkit
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  4. Burachai Sonthayanon
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Correspondence to Apinunt Udomkit.

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Treerattrakool, S., Udomkit, A., Eurwilaichitr, L. et al. Expression of Biologically Active Crustacean Hyperglycemic Hormone (CHH) of Penaeus monodon in Pichia pastoris . Mar. Biotechnol. 5, 373–379 (2003). https://doi.org/10.1007/s10126-002-0078-x

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  • DOI: https://doi.org/10.1007/s10126-002-0078-x

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