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Expression of recombinant zebrafish follicle-stimulating hormone (FSH) in methylotropic yeast Pichia pastoris

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Abstract

Pituitary gonadotropin follicle-stimulating hormone (FSH) was identified in fish two decades ago, but its functional importance in fish reproduction remains poorly defined, especially in non-salmonid species. This gap in our knowledge is partially due to the lack of the hormone in pure form in most of the species studied. We describe here the production of two different forms of biologically active recombinant zebrafish FSH (zfFSH and zfFSHHIS) using methylotrophic yeast, Pichia pastoris, as the bioreactor. One form (zfFSH) was produced as the molecule closer to the native form, with the two subunits (Cga and Fshb) expressed separately under different promoters. The other form (zfFSHHIS) was produced as a single polypeptide, with the cDNAs for the two subunits joined to form a fusion gene that contained a 6X His tag as part of the linker between the two subunits. The culture conditions were optimized for pH and incubation time for maximal production of the proteins. Using a zebrafish FSH receptor (Fshr)-based reporter gene assay, we tested and compared the biological activities of the two forms of recombinant zebrafish FSH. Our results provide useful information for the future production of recombinant gonadotropins in other fish species.

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References

  • Aizen J, Kasuto H, Golan M, Zakay H, Levavi-Sivan B (2007) Tilapia follicle-stimulating hormone (FSH): immunochemistry, stimulation by gonadotropin-releasing hormone, and effect of biologically active recombinant FSH on steroid secretion. Biol Reprod 76:692–700. doi:10.1095/biolreprod.106.055822

    Article  CAS  PubMed  Google Scholar 

  • Breton B, Govoroun M, Mikolajczyk T (1998) GTH I and GTH II secretion profiles during the reproductive cycle in female rainbow trout: Relationship with pituitary responsiveness to GnRH-A stimulation. Gen Comp Endocrinol 111:38–50. doi:10.1006/gcen.1998.7088

    Article  CAS  PubMed  Google Scholar 

  • Cui M, Li W, Liu W, Yang K, Pang Y, Haoran L (2007) Production of recombinant orange-spotted grouper (Epinephelus coioides) luteinizing hormone in insect cells by the baculovirus expression system and its biological effect. Biol Reprod 76:74–84. doi:10.1095/biolreprod.105.050484

    Article  CAS  PubMed  Google Scholar 

  • Elizur A, Zmora N, Rosenfeld H, Meiri I, Hassin S, Gordin H et al (1996) Gonadotropins β-GtHI and β-GtHII from the gilthead seabream, Sparus aurata. Gen Comp Endocrinol 102:39–46. doi:10.1006/gcen.1996.0044

    Article  CAS  PubMed  Google Scholar 

  • Fan QR, Hendrickson WA (2005) Structure of human follicle-stimulating hormone in complex with its receptor. Nature 433:269–277. doi:10.1038/nature03206

    Article  CAS  PubMed  Google Scholar 

  • Fidler AE, Lin JS, Lun S, Ng Chie W, Western A, Stent V et al (2003) Production of biologically active tethered ovine FSHβα by the methylotrophic yeast Pichia pastoris. J Mol Endocrinol 30:213–225. doi:10.1677/jme.0.0300213

    Article  CAS  PubMed  Google Scholar 

  • Ge W (2005) Intrafollicular paracrine communication in the zebrafish ovary: the state of the art of an emerging model for the study of vertebrate folliculogenesis. Mol Cell Endocrinol 237:1–10. doi:10.1016/j.mce.2005.03.012

    Article  CAS  PubMed  Google Scholar 

  • Gharib SD, Wierman ME, Shupnik MA, Chin WW (1990) Molecular biology of the pituitary gonadotropins. Endocr Rev 11:177–199

    Article  CAS  PubMed  Google Scholar 

  • Gomez JM, Weil C, Ollitrault M, Le Bail PY, Breton B, Le Gac F (1999) Growth hormone (GH) and gonadotropin subunit gene expression and pituitary and plasma changes during spermatogenesis and oogenesis in rainbow trout (Oncorhynchus mykiss). Gen Comp Endocrinol 113:413–428. doi:10.1006/gcen.1998.7222

    Article  CAS  PubMed  Google Scholar 

  • Han YS, Liao IC, Huang YS, Tzeng WN, Yu JYL (2003) Profiles of PGH-α, GTH I-β, and GTH II-β mRNA transcript levels at different ovarian stages in the wild female Japanese eel Anguilla japonica. Gen Comp Endocrinol 133:8–16. doi:10.1016/S0016-6480(03)00143-6

    Article  CAS  PubMed  Google Scholar 

  • Hayakawa Y, Morita T, Kitamura W, Kanda S, Banba A, Nagaya H et al (2008) Biological activities of single-chain goldfish follicle-stimulating hormone and luteinizing hormone. Aquaculture 274:408–415. doi:10.1016/j.aquaculture.2007.12.002

    Article  CAS  Google Scholar 

  • Hollenberg CP, Gellissen G (1997) Production of recombinant proteins by methylotrophic yeasts. Curr Opin Biotechnol 8:554–560. doi:10.1016/S0958-1669(97)80028-6

    Article  CAS  PubMed  Google Scholar 

  • Itoh H, Suzuki K, Kawauchi H (1990) The complete amino acid sequences of α subunits of chum salmon gonadotropins. Gen Comp Endocrinol 78:56–65. doi:10.1016/0016-6480(90)90047-P

    Article  CAS  PubMed  Google Scholar 

  • Jackson K, Goldberg D, Ofir M, Abraham M, Degani G (1999) Blue gourami (Trichogaster trichopterus) gonadotropic β subunits (I and II) cDNA sequences and expression during oogenesis. J Mol Endocrinol 23:177–187. doi:10.1677/jme.0.0230177

    Article  CAS  PubMed  Google Scholar 

  • Kajimura S, Yoshiura Y, Suzuki M, Aida K (2001) cDNA cloning of two gonadotropin β subunits (GTH-Iβ and -IIβ) and their expression profiles during gametogenesis in the Japanese flounder (Paralichthys olivaceus). Gen Comp Endocrinol 122:117–129. doi:10.1006/gcen.2000.7610

    Article  CAS  PubMed  Google Scholar 

  • Kamei H, Ohira T, Yoshiura Y, Uchida N, Nagasawa H, Aida K (2003) Expression of a biologically active recombinant follicle stimulating hormone of Japanese eel Anguilla japonica using methylotropic yeast, Pichia pastoris. Gen Comp Endocrinol 134:244–254. doi:10.1016/S0016-6480(03)00259-4

    Article  CAS  PubMed  Google Scholar 

  • Kasuto H, Levavi-Sivan B (2005) Production of biologically active tethered tilapia LHβα by the methylotrophic yeast Pichia pastoris. Gen Comp Endocrinol 140:222–232. doi:10.1016/j.ygcen.2004.10.016

    Article  CAS  PubMed  Google Scholar 

  • Ko H, Park W, Kim D-J, Kobayashi M, Sohn YC (2007) Biological activities of recombinant Manchurian trout FSH and LH: their receptor specificity, steroidogenic and vitellogenic potencies. J Mol Endocrinol 38:99–111. doi:10.1677/jme.1.02163

    Article  CAS  PubMed  Google Scholar 

  • Kobayashi M, Morita T, Ikeguchi K, Yoshizaki G, Suzuki T, Watabe S (2006) In vivo biological activity of recombinant goldfish gonadotropins produced by baculovirus in silkworm larvae. Aquaculture 256:433–442. doi:10.1016/j.aquaculture.2006.01.016

    Article  CAS  Google Scholar 

  • Kwok HF, So WK, Wang Y, Ge W (2005) Zebrafish gonadotropins and their receptors: I Cloning and characterization of zebrafish follicle-stimulating hormone and luteinizing hormone receptors—evidence for their distinct functions in follicle development. Biol Reprod 72:1370–1381. doi:10.1095/biolreprod.104.038190

    Article  CAS  PubMed  Google Scholar 

  • Laan M, Richmond H, He C, Campbell RK (2002) Zebrafish as a model for vertebrate reproduction: characterization of the first functional zebrafish (Danio rerio) gonadotropin receptor. Gen Comp Endocrinol 125:349–364. doi:10.1006/gcen.2001.7738

    Article  CAS  PubMed  Google Scholar 

  • Li MD, Ford JJ (1998) A comprehensive evolutionary analysis based on nucleotide and amino acid sequences of the α- and β-subunits of glycoprotein hormone gene family. J Endocrinol 156:529–542. doi:10.1677/joe.0.1560529

    Article  CAS  PubMed  Google Scholar 

  • Li YH, Bai JJ, Jian Q, Ye X, Lao HH, Li XH et al (2003) Expression of common carp growth hormone in the yeast Pichia pastoris and growth stimulation of juvenile tilapia (Oreochromis niloticus). Aquaculture 216:329–341. doi:10.1016/S0044-8486(02)00406-4

    Article  CAS  Google Scholar 

  • Mateos J, Mananos E, Martinez-Rodriguez G, Carrillo M, Querat B, Zanuy S (2003) Molecular characterization of sea bass gonadotropin subunits (α, FSHβ, and LHβ) and their expression during the reproductive cycle. Gen Comp Endocrinol 133:216–232. doi:10.1016/S0016-6480(03)00164-3

    Article  CAS  PubMed  Google Scholar 

  • Meiri I, Knibb WR, Zohar Y, Elizur A (2004) Temporal profile of β follicle-stimulating hormone, β luteinizing hormone, and growth hormone gene expression in the protandrous hermaphrodite, gilthead seabream, Sparus aurata. Gen Comp Endocrinol 137:288–299. doi:10.1016/j.ygcen.2004.04.002

    Article  CAS  PubMed  Google Scholar 

  • Melamed P, Gur G, Rosenfeld H, Elizur A, Schulz RW, Yaron Z (2000) Reproductive development of male and female tilapia hybrids (Oreochromis niloticus × O aureus) and changes in mRNA levels of gonadotropin (GtH) Iβ and IIβ subunits. J Exp Zool 286:64–75. doi:10.1002/(SICI)1097-010X(20000101)286:1<64::AID-JEZ6>3.0.CO;2-D

  • Querat B, Sellouk A, Salmon C (2000) Phylogenetic analysis of the vertebrate glycoprotein hormone family including new sequences of sturgeon (Acipenser baeri) β subunits of the two gonadotropins and the thyroid-stimulating hormone. Biol Reprod 63:222–228. doi:10.1095/biolreprod63.1.222

    Article  CAS  PubMed  Google Scholar 

  • Richard F, Robert P, Remy JJ, Martinat N, Bidart JM, Salesse R et al (1998) High-level secretion of biologically active recombinant porcine follicle-stimulating hormone by the methylotrophic yeast Pichia pastoris. Biochem Biophys Res Commun 245:847–852. doi:10.1006/bbrc.1998.8532

    Article  CAS  PubMed  Google Scholar 

  • Samaddar M, Catterall JF, Dighe RR (1997) Expression of biologically active β subunit of bovine follicle-stimulating hormone in the methylotrophic yeast Pichia pastoris. Protein Expr Purif 10:345–355. doi:10.1006/prep.1997.0745

    Article  CAS  PubMed  Google Scholar 

  • Sen Gupta C, Dighe RR (1999) Hyperexpression of biologically active human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris. J Mol Endocrinol 22:273–283. doi:10.1677/jme.0.0220273

    Article  CAS  PubMed  Google Scholar 

  • So WK, Kwok HF, Ge W (2005) Zebrafish gonadotropins and their receptors: II Cloning and characterization of zebrafish follicle-stimulating hormone and luteinizing hormone subunits—their spatial-temporal expression patterns and receptor specificity. Biol Reprod 72:1382–1396. doi:10.1095/biolreprod.104.038216

    Article  CAS  PubMed  Google Scholar 

  • Sohn YC, Yoshiura Y, Kobayashi M, Aida K (1999) Seasonal changes in mRNA levels of gonadotropin and thyrotropin subunits in the goldfish, Carassius auratus. Gen Comp Endocrinol 113:436–444. doi:10.1006/gcen.1998.7224

    Article  CAS  PubMed  Google Scholar 

  • Suetake H, Okubo K, Sato N, Yoshiura Y, Suzuki Y, Aida K (2002) Differential expression of two gonadotropin (GTH) β subunit genes during ovarian maturation induced by repeated injection of salmon GTH in the Japanese eel Anguilla japonica. Fish Sci 68:290–298. doi:10.1046/j.1444-2906.2002.00424.x

    Article  CAS  Google Scholar 

  • Suzuki K, Kawauchi H, Nagahama Y (1988) Isolation and characterization of two distinct gonadotropins from chum salmon pituitary glands. Gen Comp Endocrinol 71:292–301. doi:10.1016/0016-6480(88)90257-2

    Article  CAS  PubMed  Google Scholar 

  • Swanson P, Suzuki K, Kawauchi H, Dickhoff WW (1991) Isolation and characterization of two coho salmon gonadotropins, GTH I and GTH II. Biol Reprod 44:29–38. doi:10.1095/biolreprod44.1.29

    Article  CAS  PubMed  Google Scholar 

  • Utoh T, Horie N, Mikawa N, Okamura A, Yamada Y, Akazawa A et al (2005) Annual changes in ovarian development and plasma estradiol-17β level in reared female common Japanese conger, Conger myriaster. Fish Sci 71:38–47. doi:10.1111/j.1444-2906.2005.00928.x

    Article  CAS  Google Scholar 

  • Vischer HF, Granneman JC, Linskens MH, Schulz RW, Bogerd J (2003) Both recombinant African catfish LH and FSH are able to activate the African catfish FSH receptor. J Mol Endocrinol 31:133–140. doi:10.1677/jme.0.0310133

    Article  CAS  PubMed  Google Scholar 

  • Wong TT, Zohar Y (2004) Novel expression of gonadotropin subunit genes in oocytes of the gilthead seabream (Sparus aurata). Endocrinology 145:5210–5220. doi:10.1210/en.2004-0558

    Article  CAS  PubMed  Google Scholar 

  • Zmora N, Kazeto Y, Kumar RS, Schulz RW, Trant JM (2007) Production of recombinant channel catfish (Ictalurus punctatus) FSH and LH in S2 Drosophila cell line and an indication of their different actions. J Endocrinol 194:407–416. doi:10.1677/JOE-07-0171

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The work was substantially supported by grants (CUHK4422/04 M, CUHK4578/05 M and CUHK4587/06) from the Research Grants Council of the Hong Kong Special Administrative Region to W. Ge.

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Authors and Affiliations

  1. Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China

    Xiaobin Yu, Sze-Wah Lin & Wei Ge

  2. Department of Biology, Division of Natural Sciences, International Christian University, Tokyo, Japan

    Makito Kobayashi

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  1. Xiaobin Yu
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  2. Sze-Wah Lin
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  3. Makito Kobayashi
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  4. Wei Ge
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Correspondence to Wei Ge.

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Yu, X., Lin, SW., Kobayashi, M. et al. Expression of recombinant zebrafish follicle-stimulating hormone (FSH) in methylotropic yeast Pichia pastoris . Fish Physiol Biochem 36, 273–281 (2010). https://doi.org/10.1007/s10695-008-9244-z

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  • DOI: https://doi.org/10.1007/s10695-008-9244-z

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