Treerattrakool Supattra, Udomkit Apinunt, Eurwilaichitr Lily, Sonthayanon Burachai, Panyim Sakol
Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakhon Pathom 73170, Thailand.
Mar Biotechnol (NY). 2003 Jul-Aug;5(4):373-9. doi: 10.1007/s10126-002-0078-x.
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 alpha-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 micro g/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.
甲壳动物高血糖激素（CHH）、蜕皮抑制激素（MIH）和性腺抑制激素（GIH）是甲壳动物眼柄中X器官-窦腺复合体产生的一个主要肽家族的成员。这个肽家族在控制多种生理过程，如生长和繁殖调节中发挥重要作用。在本研究中，编码与黑虎虾斑节对虾CHH/MIH/GIH家族相关的一种肽（即Pem-CMG）的互补DNA在AOX1启动子的控制下在毕赤酵母中成功表达。重组Pem-CMG利用酿酒酵母的α-因子信号序列分泌到培养基中，没有Glu-Ala-Glu-Ala间隔肽。通过氨基末端肽测序证明重组Pem-CMG的氨基末端被正确加工。重组Pem-CMG通过反相高效液相色谱法纯化，并用于CHH活性的生物测定。纯化后重组Pem-CMG的最终产量为每升培养基260微克。从毕赤酵母产生的粗制和纯化重组Pem-CMG都显示出能够提高去眼柄斑节对虾血淋巴中的葡萄糖水平，这表明Pem-CMG肽在斑节对虾中发挥高血糖激素的作用。
