Molecular, Cellular and Animal Technology Program, Institut Pasteur de Montevideo, Montevideo, Uruguay.
Laboratory Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Montevideo, Uruguay.
Microb Biotechnol. 2024 Apr;17(4):e14444. doi: 10.1111/1751-7915.14444.
Assisted reproductive techniques are routinely used in livestock species to increase and enhance productivity. Ovarian hyperstimulation is a process that currently relies on administering pituitary-derived follicle-stimulating hormone (FSH) or equine chorionic gonadotropin in combination with other hormones to promote the maturation of multiple follicles and thereby achieve superovulation. The use of partially purified preparations of FSH extracted from natural sources is associated with suboptimal and variable results. Recombinant FSH (rFSH) has been produced in a variety of heterologous organisms. However, attaining a bioactive rFSH of high quality and at low cost for use in livestock remains challenging. Here we report the production and characterization of a single chain bovine rFSH consisting of the β- and α-subunit fused by a polypeptide linker (scbFSH) using Leishmania tarentolae as heterologous expression system. This unicellular eukaryote is non-pathogenic to mammals, can be grown in bioreactors using simple and inexpensive semisynthetic media at 26°C and does not require CO or bovine serum supplementation. Stable cell lines expressing scbFSH in an inducible fashion were generated and characterized for their productivity. Different culture conditions and purification procedures were evaluated, and the recombinant product was biochemically and biologically characterized, including bioassays in an animal model. The results demonstrate that L. tarentolae is a suitable host for producing a homogeneous, glycosylated and biologically active form of scbFSH with a reasonable yield.
辅助生殖技术在畜牧业中被常规用于提高和增强生产力。卵巢过度刺激是一个目前依赖于给予垂体来源的卵泡刺激素（FSH）或马绒毛膜促性腺激素与其他激素联合使用以促进多个卵泡成熟并由此实现超排卵的过程。使用从天然来源提取的部分纯化的 FSH 制剂与不理想和可变的结果相关联。重组 FSH（rFSH）已经在各种异源生物体中产生。然而，对于用于畜牧业的高质量和低成本的生物活性 rFSH 的获得仍然具有挑战性。在这里，我们报告了使用 Leishmania tarentolae 作为异源表达系统生产和表征由β-和α-亚基通过多肽接头融合而成的单链牛 rFSH（scbFSH）。这种单细胞真核生物对哺乳动物无致病性，能够在使用简单且廉价的半合成培养基的生物反应器中在 26°C 下生长，并且不需要 CO 或牛血清补充。以可诱导的方式表达 scbFSH 的稳定细胞系被生成并对其生产力进行了表征。评估了不同的培养条件和纯化程序，并且对重组产物进行了生化和生物学表征，包括在动物模型中的生物测定。结果表明，L. tarentolae 是生产具有合理产量的同质、糖基化和生物活性形式的 scbFSH 的合适宿主。
