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Design, production and purification of a novel recombinant gonadotropin-releasing hormone associated peptide as a spawning inducing agent for fish.
Protein Expr Purif 2020; 166:105510PE

Abstract

GnRH is a neuropeptide known to regulate reproduction in vertebrates. The purpose of this study was to design and produce recombinant gonadotropin-releasing hormone associated peptide (rGnRH/GAP) as an alternative of the previous GnRHs and native extracted hormone from tissue, to induce final maturation in fish. Decapeptide as well as GAP area sequences were compared between GnRH1, GnRH2, and mGnRH from Acipenser sp and Huso huso, respectively. Considering the conserved amino acids and the replacement of un-stable amino acids with those that were more stable against proteolytic digestion as well as had a longer half-life, the sequence was designed. The sequences of decapeptide and GAP region were synthesized and then cloned on pET28a expression vector and transformed into expression host Escherichia coli BL21(DE3). The supernatant of cultured recombinant bacteria was used for purification using TALON Metal affinity resin. The purity of the GnRH/GAP was confirmed by single 8 kDa band on SDS-PAGE and Western blot. Bioinformatics studies were performed for evaluation of homology between GnRH protein sequences and prediction of 3D protein structure using Swiss Model. The result showed that the structure prediction of the recombinant GnRH decapeptide was relatively similar to decapeptide of GnRH2 from Beluga (Huso huso). The GAP structure was similar to GAP1 of Nile tilapia (Oreochromis niloticus) and sturgeon and GnRH2 of Chinese sturgeon (Acipenser sinensis). The mass analysis showed that the sequence was exactly the same as designated sequence. Biology activity of rGnRH/GAP was tested in mature goldfish (Carassius auratus) and results showed that rGnRH/GAP had a positive effect in final maturation. Indeed 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) was increased 17 h and 24 h after injection with rGnRH/GAP and spawning stemmed from that injection. These novel findings introduce the potential of utilizing rGnRH/GAP in aquaculture.

Authors+Show Affiliations

Fisheries Department, Faculty of Animal Sciences and Fisheries, Sari Agricultural Sciences and Natural Resources University, Sari, Iran.Department of Basic Sciences, Sari Agricultural Sciences and Natural Resources University, Sari, Iran.Fisheries Department, Faculty of Animal Sciences and Fisheries, Sari Agricultural Sciences and Natural Resources University, Sari, Iran. Electronic address: skyeganeh@gmail.com.Fisheries Department, Faculty of Natural Resources, University of Guilan, Sowmeh Sara, Guilan, Iran.University of Lorraine, Unit of Animal Research and Functionalities of Animal Products, USC INRA 340, F-54505, Vandoeuvre-lès-Nancy, France.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31628987

Citation

Mohammadzadeh, Sedigheh, et al. "Design, Production and Purification of a Novel Recombinant Gonadotropin-releasing Hormone Associated Peptide as a Spawning Inducing Agent for Fish." Protein Expression and Purification, vol. 166, 2020, p. 105510.
Mohammadzadeh S, Moradian F, Yeganeh S, et al. Design, production and purification of a novel recombinant gonadotropin-releasing hormone associated peptide as a spawning inducing agent for fish. Protein Expr Purif. 2020;166:105510.
Mohammadzadeh, S., Moradian, F., Yeganeh, S., Falahatkar, B., & Milla, S. (2020). Design, production and purification of a novel recombinant gonadotropin-releasing hormone associated peptide as a spawning inducing agent for fish. Protein Expression and Purification, 166, p. 105510. doi:10.1016/j.pep.2019.105510.
Mohammadzadeh S, et al. Design, Production and Purification of a Novel Recombinant Gonadotropin-releasing Hormone Associated Peptide as a Spawning Inducing Agent for Fish. Protein Expr Purif. 2020;166:105510. PubMed PMID: 31628987.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Design, production and purification of a novel recombinant gonadotropin-releasing hormone associated peptide as a spawning inducing agent for fish. AU - Mohammadzadeh,Sedigheh, AU - Moradian,Fatemeh, AU - Yeganeh,Sakineh, AU - Falahatkar,Bahram, AU - Milla,Sylvain, Y1 - 2019/10/16/ PY - 2019/06/28/received PY - 2019/08/10/revised PY - 2019/10/13/accepted PY - 2019/10/20/pubmed PY - 2019/10/20/medline PY - 2019/10/20/entrez KW - Decapeptide KW - GAP KW - Half-life KW - Proteolytic stability KW - rGnRH SP - 105510 EP - 105510 JF - Protein expression and purification JO - Protein Expr. Purif. VL - 166 N2 - GnRH is a neuropeptide known to regulate reproduction in vertebrates. The purpose of this study was to design and produce recombinant gonadotropin-releasing hormone associated peptide (rGnRH/GAP) as an alternative of the previous GnRHs and native extracted hormone from tissue, to induce final maturation in fish. Decapeptide as well as GAP area sequences were compared between GnRH1, GnRH2, and mGnRH from Acipenser sp and Huso huso, respectively. Considering the conserved amino acids and the replacement of un-stable amino acids with those that were more stable against proteolytic digestion as well as had a longer half-life, the sequence was designed. The sequences of decapeptide and GAP region were synthesized and then cloned on pET28a expression vector and transformed into expression host Escherichia coli BL21(DE3). The supernatant of cultured recombinant bacteria was used for purification using TALON Metal affinity resin. The purity of the GnRH/GAP was confirmed by single 8 kDa band on SDS-PAGE and Western blot. Bioinformatics studies were performed for evaluation of homology between GnRH protein sequences and prediction of 3D protein structure using Swiss Model. The result showed that the structure prediction of the recombinant GnRH decapeptide was relatively similar to decapeptide of GnRH2 from Beluga (Huso huso). The GAP structure was similar to GAP1 of Nile tilapia (Oreochromis niloticus) and sturgeon and GnRH2 of Chinese sturgeon (Acipenser sinensis). The mass analysis showed that the sequence was exactly the same as designated sequence. Biology activity of rGnRH/GAP was tested in mature goldfish (Carassius auratus) and results showed that rGnRH/GAP had a positive effect in final maturation. Indeed 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) was increased 17 h and 24 h after injection with rGnRH/GAP and spawning stemmed from that injection. These novel findings introduce the potential of utilizing rGnRH/GAP in aquaculture. SN - 1096-0279 UR - https://www.unboundmedicine.com/medline/citation/31628987/Design,_production_and_purification_of_a_novel_recombinant_gonadotropin-releasing_hormone_associated_peptide_as_a_spawning_inducing_agent_for_fish L2 - https://linkinghub.elsevier.com/retrieve/pii/S1046-5928(19)30345-6 DB - PRIME DP - Unbound Medicine ER -