Identification and Soluble Expression of Recombinant Proteins of fshβ in Conger myriaster

The Conger myriaster is commercially important in fisheries and aquaculture. The artificial culture of C. myriaster is presently conducted by catching wild fry. Recently, this resource has notably declined, highlighting an urgent need to develop seedling production for its aquaculture. C. myriaster gonads cannot mature naturally in artificial culture owing to insufficient synthesis of endogenous gonadotropins. However, their gonads can be induced to mature by injecting hormones in vivo. The follicle-stimulating hormone (FSH) gene is crucial in fish sexual development and reproduction, and its regulatory mechanisms have been extensively studied. In this study, the coding sequence (CDS) of fshβ was cloned by PCR techniques. Bioinformatical analysis revealed that the CDS of FSH comprises 378 bp, encoding 125 amino acids. By analyzing the physical and chemical properties of the protein, the fshβ protein has a molecular weight of 13.19 kDa. The aliphatic index was 71.04, and the theoretical pI was 6.00. An instability index value of 51.57 indicates a state of instability. The functional domain analysis showed that the fshβ protein has a highly conserved GHB functional domain (comprising 104 amino acids at 19–122). The hydrophilicity and hydrophobicity analysis revealed that the fshβ protein is a hydrophilic protein. The 6th and 7th amino acids (Ala) and 8th amino acid (Leu) exhibited the strongest hydrophobicity (2.178), whereas the 106th amino acid (Asn) exhibited the strongest hydrophilicity (–1.722). The leader peptide and transmembrane domain analyses of fshβ protein revealed that the 1–19 amino acids are signal peptides without any transmembrane domains. The analysis of protein glycosylation sites showed that the fshβ protein contains two N-glycosylation sites and three O-glycosylation sites. The analysis of protein phosphorylation sites demonstrated that the fshβ protein contains 14 potential phosphorylation sites. Specifically, seven serine (Ser) and seven tyrosine (Tyr) phosphorylation sites exist. Subcellular localization analysis revealed that the fshβ protein was mainly localized in the nucleus. The secondary structure of the fshβ protein mainly comprises an alpha helix (12.0%), extended strand (24.8%), and random coil (63.2%). The tertiary structure of C. myriaster fshβ protein was compared with that of Anguilla japonica, A. rostrata, A. Anguilla, A. marmorata and Homo sapiens. The C. myriaster fshβ protein tertiary structure was observed to be similar to that of A. Japanica, A. rostrata, A. Anguilla, A. marmorata. However, differences were observed with the tertiary structure of H. sapiens. Based on the sequence alignment and phylogenetic analysis, the C. myriaster fshβ exhibited a closer evolutionary relationship with A. japonica, A. rostrata, A. anguilla and A. marmorata. The fshβ amino acid sequence of C. myriaster compared with A. japonica, A. rostrata, A. anguilla, and A. marmorata revealed similarities of 70.08%, 70.08%, 70.08% and 68.50%, respectively. The fshβ of C. myriaster exhibits high homology with other eels but low homology with other bony fish and mammals, indicating that fshβ has evolutionary differences during evolution. This study presents the successful production of recombinant C. myriaster fshβ protein using the pET-28a(+) expression system. NdeⅠ and XhoⅠ cleavage sites were inserted at both ends of the primers, and then fshβ was amplified by PCR. Subsequently, the fragment was conjugated to the pEASY-T1 vector to obtain the recombined plasmids, named pEASY-T1-fshβ. The recombinant plasmid pEASY-T1-fshβ and the pET-28a(+) vector were double-digested using restriction endonuclease NdeⅠ/XhoⅠ. After double-digested, the fragment was ligated into the pET-28a(+) vector to obtain the recombined plasmids named pET-28a-fshβ. To investigate the biological activities and physiological significance of pET-28a-fshβ, we used the pET protein fusion and purification system to produce plasmids pET-28a-fshβ in Trans1-T1 competent cells. Recombinant plasmids pET-28a-fshβ were transferred into Rosetta (DE3) cells, which were cultured under different IPTG induction conditions (0.01, 0.1, 0.5, and 1 mmol/L) at a temperature of 16 ℃ for 16 h. fshβ was efficiently expressed at different IPTG concentrations and under the above induction conditions. Therefore, the following optimized parameters were used in subsequent experiments: IPTG concentration of 0.5 mmol/L, incubated at 16 ℃ for 16 h. The supernatant and precipitate were analyzed using SDS-PAGE following induction of expression. SDS-PAGE analysis showed an evident thickened band at 26.0 kDa. It was also present in the supernatant as a soluble protein. The protein was purified using a His-tagged nickel affinity chromatography column. Western blot analysis confirmed the presence of the purified protein, demonstrating that the recombinant protein was specifically recognized by antibodies. The molecular mass of the protein was approximately 26.0 kDa, which was consistent with the expected size. This result indicates that the fshβ recombinant protein was successfully expressed. The results of this study revealed the preparation of recombinant fshβ protein in the C. myriaster to provide a theoretical reference for the subsequent use of exogenous recombinant fshβ protein to induce sexual maturation in Parent fish.