Production of recombinant orange-spotted grouper (Epinephelus coioides) follicle-stimulating hormone (FSH) in single-chain form and dimer form by Pichia pastoris and their biological activities

被引：29
作者：
Chen, Jun [1 ,2 ]
Zhang, Yanhong [1 ,2 ]
Tang, Zhiguo [1 ,2 ]
Mao, Jiewei [1 ,2 ]
Kuang, Zhonglei [1 ,2 ]
Qin, Chaobin [1 ,2 ]
Li, Wensheng [1 ,2 ]
机构：
[1] Sun Yat Sen Univ, State Key Lab Bioconttol, Inst Aquat Econ Anim, Guangzhou 510275, Guangdong, Peoples R China
[2] Sun Yat Sen Univ, Guangdong Prov Key Lab Aquat Econ Anim, Guangzhou 510275, Guangdong, Peoples R China
来源：
GENERAL AND COMPARATIVE ENDOCRINOLOGY | 2012年 / 178卷 / 02期
基金：
中国国家自然科学基金;
关键词：
Follicle-stimulating hormone; Pichia pastoris; Steroidogenesis; Early ovarian development; Epinephelus coioides; GONADOTROPIN-RELEASING-HORMONE; MESSENGER-RNA EXPRESSION; LUTEINIZING-HORMONE; CHORIONIC-GONADOTROPIN; GLYCOPROTEIN HORMONES; GENE-EXPRESSION; JAPANESE EEL; LH-BETA; OVARIAN STEROIDOGENESIS; ANGUILLA-JAPONICA;
D O I：
10.1016/j.ygcen.2012.05.009
中图分类号：
R5 [内科学];
学科分类号：
1002 ; 100201 ;
摘要：
FSH is a key regulator of steroidogenesis and gonadal growth in teleosts. However, function of FSH is elusive in grouper due to the lack of purified and native FSH. In the present study, we reported production of bioactive orange-spotted grouper (Epinephelus coioides) FSH in dimer form and single-chain form by Pichia pastoris. Dimer form of recombinant grouper FSH (rgFSHba) was accomplished by co-expressing mature FSHb-subunit and a-subunit genes. Fusion of mature FSHb-subunit and a-subunit genes together linking with a polypeptide (4x(Gly-Ser)-Gly-Thr) gene generated single-chain form of recombinant grouper FSH (rgFSHb-a). Recombinant grouper common alpha-subunit (rgCga) and FSHb-subunit (rgFSHb) were also separately produced. Recombinant proteins were verified by Western blot and mass spectrometry assays, and characterized by deglycosylation analysis. Deglycosylation assay suggested that glycosylation of recombinant FSH mainly occurred on common a-subunit. Bioactivities of recombinant proteins were initially evaluated by activating grouper FSH receptor, and further demonstrated by incubating ovarian fragments of adult grouper and intraperitoneal injection in juvenile female grouper. Two forms of recombinant FSH presented similar biological activities of activating FSH receptor and stimulating in vitro testosterone (T) and estradiol-17 beta (E2) secretion, though the dimer form functioned slightly weaker than the single-chain form. However, injections of rgFSHb-a or rgFSHba could significantly increase serum T and E2 levels, induce early ovarian development, reduce hypothalamic gnrh 1 mRNA level, and increase hypothalamic cyp19a1b mRNA level. Data in this study suggested that recombinant gonadotropin could be produced in dimer form or single-chain form by P. pastoris, and FSH could regulate steroidogenesis and early ovarian development in juvenile grouper. (C) 2012 Elsevier Inc. All rights reserved.
引用
收藏
页码：237 / 249
页数：13
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