Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, Microbiological Staff Room, College of Life Sciences, South China Agricultural University, Wushan Road, Tianhe District, Guangzhou, 510642, Guangdong, China.
Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, Guangdong, 510642, China.
Microb Cell Fact. 2020 Feb 18;19(1):35. doi: 10.1186/s12934-020-01304-5.
Porcine growth hormone (pGH) is a class of peptide hormones secreted from the pituitary gland, which can significantly improve growth and feed utilization of pigs. However, it is unstable and volatile in vitro. It needs to be encapsulated in liposomes when feeding livestock, whose high cost greatly limits its application in pig industry. Therefore we attempted to express pGH as intracellular soluble protein in Pichia pastoris and feed these yeasts with partial wall-breaking for swine, which could release directly pGH in intestine tract in case of being degraded in intestinal tract with low cost. In order to improve the intracellular soluble expression of pGH protein in Pichia pastoris and stability in vitro, we optimized the pGH gene, and screened molecular chaperones from E. coli and Pichia pastoris respectively for co-expressing with pGH. In addition, we had also explored conditions of mechanical crushing and fermentation. The results showed that the expression of intracellular soluble pGH protein was significantly increased after gene optimized and co-expressed with Ssa1-Sis1 chaperone from Pichia pastoris. Meanwhile, the optimal conditions of partial wall-breaking and fermentation of Pichia pastoris were confirmed, the data showed that the intracellular expression of the optimized pGH protein co-expressed with Ssa1-Sis1 could reach 340 mg/L with optimal conditions of partial wall-breaking and fermentation. Animal experiments verified that the optimized pGH protein co-expression with Ssa1-Sis1 had the best promoting effects on the growth of piglets. Our study demonstrated that Ssa1-Sis1 could enhance the intracellular soluble expression of pGH protein in Pichia pastoris and that partial wall-breaking of yeast could prevent pGH from degradation in vitro, release targetedly in the intestine and play its biological function effectively. Our study could provide a new idea to cut the cost effectively, establishing a theoretical basis for the clinic application of unstable substances in vitro.
猪生长激素（pGH）是一类由垂体分泌的肽类激素，能显著提高猪的生长和饲料利用率。但它在体外不稳定且易挥发，在给家畜投喂时需要用脂质体包埋，但其高昂的成本大大限制了其在猪产业中的应用。因此，我们试图在毕赤酵母中表达 pGH 作为细胞内可溶性蛋白，并对这些酵母进行部分破壁处理，以便在猪体内以低成本直接在肠道中释放 pGH，以防其在肠道中被降解。为了提高 pGH 蛋白在毕赤酵母中的细胞内可溶性表达和体外稳定性，我们对 pGH 基因进行了优化，并分别从大肠杆菌和毕赤酵母中筛选分子伴侣与 pGH 共表达。此外，我们还探索了机械破碎和发酵条件。结果表明，基因优化后与毕赤酵母的 Ssa1-Sis1 伴侣共表达可显著提高细胞内可溶性 pGH 蛋白的表达。同时，还确定了毕赤酵母部分破壁和发酵的最佳条件，数据表明，在最佳的部分破壁和发酵条件下，与 Ssa1-Sis1 共表达的优化后的 pGH 蛋白的细胞内表达量可达到 340mg/L。动物实验验证了与 Ssa1-Sis1 共表达的优化后的 pGH 蛋白对仔猪生长具有最佳的促进作用。本研究表明，Ssa1-Sis1 可以增强 pGH 蛋白在毕赤酵母中的细胞内可溶性表达，而酵母的部分破壁可以防止 pGH 在体外降解，在肠道中靶向释放，有效地发挥其生物学功能。本研究为有效降低成本提供了新思路，为体外不稳定物质的临床应用奠定了理论基础。
