Rodríguez María Celeste, Mussio Pablo Esteban, Villarraza Javier, Tardivo María Belén, Antuña Sebastián, Fontana Diego, Ceaglio Natalia, Prieto Claudio
UNL, CONICET, FBCB (School of Biochemistry and Biological Sciences), CBL (Biotechnological Center of Litoral), Cell Culture Laboratory, Ciudad Universitaria, Ruta Nacional 168 - Km 472.4 - C.C. 242 - (S3000ZAA), Santa Fe, Argentina.
Biotecnofe S.A. PTLC, Ruta 168 Pje El Pozo, (3000), Santa Fe, Argentina.
Protein J. 2023 Feb;42(1):24-36. doi: 10.1007/s10930-023-10092-x. Epub 2023 Jan 18.
Equine chorionic gonadotropin (eCG) is a glycoprotein hormone widely used in timed artificial ovulation (TAI) and superovulation protocols to improve the reproductive performance in livestock. Until recently, the only eCG products available in the market for veterinary use consisted in partially purified preparations of pregnant mare serum gonadotropin (PMSG). Here, a bioactive recombinant eCG (reCG) produced in suspension CHO-K1 cells was purified employing different chromatographic methods (hydrophobic interaction chromatography and reverse-phase (RP)-HPLC) and compared with a RP-HPLC-purified PMSG. To gain insight into the structural and functional characteristics of reCG, a bioinformatics analysis was performed. An exhaustive characterization comprising the determination of the purity degree, aggregates and nicked forms through SDS-PAGE, RP-HPLC and SEC-HPLC was performed. Higher order structures were studied by fluorescence spectroscopy and SEC-HPLC. Isoforms profile were analyzed by isoelectric focusing. Glycosylation analysis was performed through pulsed amperometric detection and PNGase F treatment following SDS-PAGE and weak anion exchange-HPLC. Slight differences between the purified recombinant hormones were found. However, recombinant molecules and PMSG exhibited variations in the glycosylation pattern. In fact, differences in sialic acid content between two commercial preparations of PMSG were also obtained, which could lead to differences in their biological potency. These results show the importance of having a standardized production process, as occurs in a recombinant protein bioprocess. Besides, our results reflect the importance of the glycan moieties on eCG conformation and hence in its biological activity, preventing denaturing processes such as aggregation.
马绒毛膜促性腺激素（eCG）是一种糖蛋白激素，广泛应用于定时人工授精（TAI）和超数排卵方案中，以提高家畜的繁殖性能。直到最近，市场上可用于兽医用途的唯一eCG产品是孕马血清促性腺激素（PMSG）的部分纯化制剂。在此，采用不同的色谱方法（疏水相互作用色谱和反相（RP）-HPLC）对悬浮CHO-K1细胞中产生的具有生物活性的重组eCG（reCG）进行纯化，并与RP-HPLC纯化的PMSG进行比较。为了深入了解reCG的结构和功能特性，进行了生物信息学分析。通过SDS-PAGE、RP-HPLC和SEC-HPLC对纯度、聚集体和切口形式进行了详尽的表征测定。通过荧光光谱和SEC-HPLC研究了高级结构。通过等电聚焦分析了异构体谱。在SDS-PAGE和弱阴离子交换-HPLC之后，通过脉冲安培检测和PNGase F处理进行糖基化分析。发现纯化的重组激素之间存在细微差异。然而，重组分子和PMSG在糖基化模式上表现出差异。事实上，两种市售PMSG制剂之间的唾液酸含量也存在差异，这可能导致它们的生物活性不同。这些结果表明了拥有标准化生产工艺的重要性，就像重组蛋白生物工艺中那样。此外，我们的结果反映了聚糖部分对eCG构象及其生物活性的重要性，可防止诸如聚集等变性过程。
