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

Abstract

A beta actin cDNA of Tanichthys albonubes was isolated through the RT-PCR and RACE approach. The cDNA was 1,787-bp in length, including a 1,128-bp CDS, a 95-bp 5'UTR and a 564-bp 3'UTR. Genomic DNA containing the transcription region and 5'-flanking region was cloned based on the beta actin cDNA by Genome walker. A 3,000-bp beta actin gene promoter was then produced by PCR according to the sequences of the 5'-flanking region and the first intron. This promoter consisted of a 1,800-bp 5'-flanking region, and a 1,200-bp 5'-UTR. 3 transcription elements, CAAT box, CArG motif and TATA box were found in the 5'-flanking region. This promoter was inserted into the vector pDsRed2-1 and microinjected into fertilized eggs of Tanichthys albonubes to prove its transcription activity. The beta actin promoter and GH CDS of Tanichthys albonubes were then fused to construct an expression vector pTLA-GH. GH-transgenic Tanichthys albonubes was obtained by microinjection of the pTLA-GH into the fertilized eggs. Fast-growth individuals were observed in the transgenic group and the body weight of the largest individual was 2.1-fold that of the maximum in its non-transgenic siblings in 100 dph. In addition, a co-injection strategy was employed with pTLA-DsRed and pTLA-GH vector and proven to enhance the efficiency of GH-transgenic fish detection.

Follicle-Stimulating Hormone and Luteinizing Hormone Mediate the Androgenic Pathway in Leydig Cells of an Evolutionary Advanced Teleost1

“…Both recombinants were produced and secreted as singlechain polypeptides consisting of the specific b chain and the common a chain linked by the carboxyl-terminal peptide of hCG. Single-chain gonadotropins have been previously produced in different teleosts, including the Nile tilapia (Oreochromis niloticus) [48,49], Japanese eel [50], Manchurian trout [16], zebrafish [27,51], and European sea bass [18], using either the methylotrophic yeast Pichia pastoris, insect or mammalian cell lines, or baculovirus-silkworm systems, and are suggested to have a longer half-life compared with the production of separate subunits [52,53]. By adding a His tag to the protein, which does not seem to affect the bioactivity of the hormones [27,50], sole recombinant gonadotropins could be efficiently purified from the culture medium (;90%) using affinity chromatography.…”

Studies in Zebrafish Reveal Unusual Cellular Expression Patterns of Gonadotropin Receptor Messenger Ribonucleic Acids in the Testis and Unexpected Functional Differentiation of the Gonadotropins

“…Refs. 11,13,16,[72][73][74][75][76] reflect the biological activities of the pituitary hormones could be answered only by comparing highly purified gonadotropins with homologous recombinant proteins, a setting not feasible in zebrafish due to its small size. Nevertheless, different recombinant gonadotropins, including single-chain and/or N-terminal His-tagged molecules (like the hormones used in this study), have been shown to elicit biological effects comparable to heterodimeric purified native hormones, which has been explained by the receptors' capacity to specifically recognize their ligands even if those are presented in different conformations (77)(78)(79)(80)(81)(82).…”

Toward developing recombinant gonadotropin-based hormone therapies for increasing fertility in the flatfish Senegalese sole

“…In addition to CHO cells, teleost recombinant gonadotropins have been produced in different heterologous eukaryotic systems, such as the yeast Pichia pastoris [31, 32, 36, 39], the baculovirus-silkworm system [33, 37] and insect Sf9 cells [34]. However, the type of glycosylation of the hormones that occurs in mammalian systems renders the polypeptides more stable in fish plasma than those produced in other heterologous hosts [38], and can also enhance their biological activity [40, 41].…”