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.
Similar content being viewed by others
References
L. Briand V. Perez J.C. Huet E. Danty C. Masson J.C. Pernollet (1999) ArticleTitleOptimization of the production of a honeybee odorant-binding protein by Pichia pastoris. Protein Expr Purif 15 362–369 Occurrence Handle1:CAS:528:DyaK1MXhvF2ju7c%3D Occurrence Handle10092496
J.L. Cereghino J.M. Cregg (2000) ArticleTitleHeterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24 45–66 Occurrence Handle10.1016/S0168-6445(99)00029-7 Occurrence Handle1:CAS:528:DC%2BD3cXltVClug%3D%3D Occurrence Handle10640598
E.S. Chang (1997) Chemistry of crustacean hormones that regulate growth and reproduction. M. Fingerman R. Nagabhushanam M.-F. Thompson (Eds) Recent Advances in Marine Biotechnology Science Publishers Enfield, N.H. 1:163–178
E.S. Chang G.D. Prestwich M.J. Bruce (1990) ArticleTitleAmino acid sequence of peptide with both molt-inhibiting and hyperglycemic activities in the lobster, Homarus americanus. Biochem Biophys Res Commun 171 818–826 Occurrence Handle1:CAS:528:DyaK3MXlslKluw%3D%3D Occurrence Handle2169734
G. Charmantier M. Charmantier-Daures F. Van Herp (1997) Hormonal regulation of growth and reproduction in crustaceans. M. Fingerman R. Nagabhushanam M.-F. Thompson (Eds) Recent Advances in Marine Biotechnology Science Publishers Enfield, N.H. 1:109–161
Chooluck, S., Wongsombat, T., Udomkit, A., Sonthayanon, B., and Panyim, S. (2000). Expression of Pem-CMG1, an eyestalk neuropeptide of Penaeus monodon in Escherichia coli. Phuket, Thailand: Proceedings of the 2 nd National Symposium on Marine Shrimp, 56–62.
J.J. Clare M.A. Romanos F.B. Rayment I.E. Rowedder M.A. Smith M.M. Payne K. Sreekrishna C.A. Henwood (1991) ArticleTitleProduction of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. Gene 105 205–212 Occurrence Handle10.1016/0378-1119(91)90152-2 Occurrence Handle1:CAS:528:DyaK3MXmslamt78%3D Occurrence Handle1937016
J.M. Cregg (1999) Expression in the methylotrophic yeast Pichia pastoris. J.M. Fernandez J.P. Hoeffler (Eds) Gene Expression Systems Academic Press NewYork, N.Y. 157–191
J.M. Cregg T.S. Vedvick W.C. Raschke (1993) ArticleTitleRecent advances in the expression of foreign genes in Pichia pastoris. Biotechnology 11 905–910 Occurrence Handle1:CAS:528:DyaK3sXmsVajtbk%3D Occurrence Handle7763913
M.L. Davey M.R. Hall R.H. Willis R.W. Oliver M.J. Thurn K.J. Wilson (2000) ArticleTitleFive crustacean hyperglycemic family hormones of Penaeus monodon: complementary DNA sequence and identification in single sinus glands by electrospray ionization–Fourier transform mass spectrometry. Mar Biotechnol 2 80–91 Occurrence Handle1:CAS:528:DC%2BD3cXhs1Wqs70%3D Occurrence Handle10804243
D.P.V. De Kleijn E.P.H. De Leeuw M.C. Van Den Berg G.J.M. Martens F. Van Herp (1995) ArticleTitleCloning and expression of two mRNAs encoding structurally different crustacean hyperglycemic hormone precursors in the lobster Homarus americanus. Biochim Biophys Acta 1260 62–66 Occurrence Handle1:CAS:528:DyaK2MXis12hu7w%3D Occurrence Handle7999796
D.P.V. De Kleijn K.P.C. Janssen S.L. Waddy R. Hegeman W.Y. Lai G.J.M. Martens F. Van Herp (1998) ArticleTitleExpression of the crustacean hyperglycemic hormones and the gonad-inhibiting hormone during the reproductive cycle of the female American lobster Homarus americanus. J Endrocrinol 156 291–298 Occurrence Handle1:CAS:528:DyaK1cXhtV2ktrY%3D
P.-L. Gu K.L. Yu S.-M. Chan (2000) ArticleTitleMolecular characterization of an additional shrimp hyperglycemic hormone: cDNA cloning, gene organization, expression and biological assay of recombinant proteins. FEBS Lett 472 122–128 Occurrence Handle1:CAS:528:DC%2BD3cXislarsbw%3D Occurrence Handle10781818
P.-L. Gu K.H. Chu S.-M. Chan (2001) ArticleTitleBacterial expression of the shrimp molt-inhibiting hormone (MIH): antibody production, immunocytochemical study and biological assay. Cell Tissue Res 303 129–136 Occurrence Handle1:CAS:528:DC%2BD3MXisFCqtQ%3D%3D Occurrence Handle11236000
D.R. Higgings J.M. Cregg (1998) Methods in Molecular Biology. Pichia Protocols, vol 103. Humana Press Totowa, N.J.
R.S.E.W. Leuven P.P. Jaros R. Van Herp R. Keller (1982) ArticleTitleSpecies or group specificity in biological and immunological studies of crustacean hyperglycemic hormone. Gen Comp Endocrinol 46 288–296 Occurrence Handle1:CAS:528:DyaL38XhtF2mtr4%3D Occurrence Handle6175550
J. Martinez M. Rodriguez J.P. Bali J. Laur (1986) ArticleTitlePhenethyl ester derivative analogous of the C-terminal tetrapeptide of gastrin as potent gastrin antagonists. J Med Chem 29 2201–2206 Occurrence Handle1:CAS:528:DyaL28Xlslejsb8%3D Occurrence Handle3783582
T. Ohira T. Nishimura H. Sonobe A. Okuno T. Watanabe H. Nagasawa I. Kawazoe K. Aida (1999) ArticleTitleExpression of a recombinant molt-inhibiting hormone of the kuruma prawn Penaeus japonicus in Escherichia coli. Biosci Biotechnol Biochem 63 1576–1581 Occurrence Handle1:CAS:528:DyaK1MXmsVOktb8%3D Occurrence Handle10540746
R.J. Raemaekers L. de Muro J.A. Gatehouse A.P. Fordham-Skelton (1999) ArticleTitleFunctional phytohemagglutinin (PHA) and Galanthus nivalis agglutinin (GNA) expressed in the PHA-E signal peptide. Eur J Biochem 265 394–403 Occurrence Handle1:CAS:528:DyaK1MXmsFKksb0%3D Occurrence Handle10491197
W. Sithigorngul S. Jaideechoeys W. Saraithongkum S. Longyant P. Sithigorngul (1999) ArticleTitlePurification and characterization of an isoform of crustacean hyperglycemic hormone from the eyestalk of Macrobrachium rosenbergii. J Exp Zool 284 217–224 Occurrence Handle1:CAS:528:DyaK1MXksVyjsbw%3D Occurrence Handle10404650
C. Spanings-Pierrot D. Soyez F. Van Herp M. Gompel G. Skaret E. Grousset G. Charmamtier (2000) ArticleTitleInvolvement of crustacean hyperglycemic hormone in the control of gill ion transport in the crab Pachygrapsus marmorqatus. Gen Comp Endrocrinol 119 340–350 Occurrence Handle1:CAS:528:DC%2BD3cXmvFegsr0%3D
A. Udomkit S. Chooluck B. Sonthayanon S. Panyim (2000) ArticleTitleMolecular cloning of a cDNA encoding a member of CHH/MIH/GIH family from Penaeus monodon and analysis of its gene structure. J Exp Mar Biol Ecol 224 145–156
F. Van Herp (1998) Molecular, cytological and physiological aspects of the crustacean hyperglycemic hormone family. E.M. Coast S.G. Webster (Eds) Recent Advance in Arthropod Endocrinology Cambridge University Press Cambridge, U.K. 65:53–70
W.E. Van Nostrand A.H. Schmaier B.R. Neiditch R.S. Siegel W. Raschke S.S. Sisodia S.L. Eagner (1994) ArticleTitleExpression purification and characterization of the kunitz type proteinase inhibitor domain of the amyloid β-protein precursor like protien-2. Biochim Biophys Acta 1209 165–170 Occurrence Handle1:STN:280:ByqC3M%2FotVY%3D Occurrence Handle7811686
S.L. Wagner R.S. Siegel T.S. Vedvick W.C. Raschke W.E. Van Nostrand (1992) ArticleTitleHigh level expression, purification and characterization of the kunitz type protease inhibitor domain of protease nexin-2/amyloid β-protein precursor. Biochem Biophys Res Commun 186 1138–1145 Occurrence Handle1:CAS:528:DyaK38XmtVOktr8%3D Occurrence Handle1497647
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.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s10126-002-0078-x