Abstract
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.
Highlights
The porcine growth hormone is a single-chain polypeptide hormone produced by the secretion of the anterior pituitary gland [1,2,3]. pGH makes an important impact on the regulation of postnatal growth [2], effectively regulates metabolism [4] and promotes muscle growth [5].The recombinant protein was secreted into the extracellular domain or located in the cytoplasm through the Golgi body after the endoplasmic reticulum synthesis process [6]
Construction of recombinant strains of pGH gene and expression of pGH proteins By extracting the total RNA from porcine cerebellum and obtaining cDNA from reverse transcriptase, the pGH gene was obtained by pGH primer amplification
The results showed that after co-expression of Bip–PDI molecular chaperones with optimized pGH gene at 30 °C for 24 h, there were no visible pGH protein band in the cells’ supernatant (SDS–polyacrylamide gel electrophoresis (PAGE)), and the yield of insoluble pGH protein showed a decrease after induction in Pichia pastoris a2 cells (Fig. 6)
Summary
The porcine growth hormone (pGH) is a single-chain polypeptide hormone produced by the secretion of the anterior pituitary gland [1,2,3]. pGH makes an important impact on the regulation of postnatal growth [2], effectively regulates metabolism [4] and promotes muscle growth [5].The recombinant protein was secreted into the extracellular domain or located in the cytoplasm through the Golgi body after the endoplasmic reticulum synthesis process [6]. The molecular chaperone is a class of molecules that play an important role in protein translation, transport, folding and modification [8]. Ssa and Sis, play a very important role in the formation of the correct native conformation of nascent peptides. Hsp 70 (including chaperones Bip and PDI) can identify the structural characteristics of most of the initial peptide chains to help them fold properly [10]. PDI can identify disulfide bond to help the protein fold correctly, as supported by our previous observations [11]. E. coli GroE (including chaperones GroEL and GroES) could form complexes with nonnative polypeptides to avoid intermolecular aggregation formation of newly synthesized proteins [12]. Defects in prokaryotic posttranslational modification, as well as production of insoluble and non-secreted protein, limited the effectiveness of this system. The yield of pGH in these eukaryotic expression systems is too low to be applied for production and the high cost is another limitation in its application
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