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Pichia pastoris is a highly successful system for the large-scale expression of heterologous proteins, with the added capability of performing most eukaryotic post-translational modifications. However, this system has one significant disadvantage - frequent proteolytic degradation by P. pastoris proteases of heterologously expressed proteins. Several methods have been proposed to address this problem, but none has proven fully effective. We tested the effectiveness of a broad specificity protease inhibitor to control proteolysis. A recombinant variant of the BPTI-Kunitz protease inhibitor ShPI-1 isolated from the sea anemone Stichodactyla helianthus, was expressed in P. pastoris. The recombinant inhibitor (rShPI-1A), containing four additional amino acids (EAEA) at the N-terminus, was folded similarly to the natural inhibitor, as assessed by circular dichroism. rShPI-1A had broad protease specificity, inhibiting serine, aspartic, and cysteine proteases similarly to the natural inhibitor. rShPI-1A protected a model protein, recombinant human miniproinsulin (rhMPI), from proteolytic degradation during expression in P. pastoris. The addition of purified rShPI-1A at the beginning of the induction phase significantly protected rhMPI from proteolysis in culture broth. The results suggest that a broad specificity protease inhibitor such as rShPI-1A can be used to improve the yield of recombinant proteins secreted from P. pastoris., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

P ichia pastoris is a highly successful system for the large-scale expression of heterologous proteins, with the added capability of performing most eukaryotic post-translational modifications. However, this system has one significant disadvantage - frequent proteolytic degradation by P . pastoris proteases of heterologously expressed proteins. Several methods have been proposed to address this problem, but none has proven fully effective. We tested the effectiveness of a broad specificity protease inhibitor to control proteolysis. A recombinant variant of the BPTI- Kunitz protease inhibitor Sh PI-1 isolated from the sea anemone S tichodactyla helianthus, was expressed in P . pastoris. The recombinant inhibitor (r Sh PI-1 A), containing four additional amino acids ( EAEA) at the N-terminus, was folded similarly to the natural inhibitor, as assessed by circular dichroism. r Sh PI-1 A had broad protease specificity, inhibiting serine, aspartic, and cysteine proteases similarly to the natural inhibitor. r Sh PI-1 A protected a model protein, recombinant human miniproinsulin (rh MPI), from proteolytic degradation during expression in P . pastoris. The addition of purified r Sh PI-1 A at the beginning of the induction phase significantly protected rh MPI from proteolysis in culture broth. The results suggest that a broad specificity protease inhibitor such as r Sh PI-1 A can be used to improve the yield of recombinant proteins secreted from P . pastoris. [ABSTRACT FROM AUTHOR]

Background Recombinant erythropoietin (EPO) has been marketed as biopharmaceutical for anemia and chronic renal failure. Long-acting EPO variants that aimed at achieving less frequent dosing have been generated, either by the addition of glycosylation sites or increasing its molecular weight. Methods The hEPO cDNA linked to the human IgG Fc fragment was cloned as a single codifying gene on the pAdtrack-CMV vector, yielding the recombinant adenoviral genome. For in vitro and in vivo expression assays cervical cancer cell line (SiHa) and nulliparous goats were used, respectively. The hematopoietic activity of EPO-Fc, expressed as the differential increment of hematocrit was evaluated in B6D2F1 mice. NP-HPLC of the 2AB-labeled N-glycan was carried out to profile analysis. Results The direct transduction of mammary secretory cells with adenoviral vector is a robust methodology to obtain high levels of EPO of up to 3.5 mg/mL in goat's milk. SiHa-derived EPO-Fc showed significant improvement in hematopoietic activity compared to the commercial hEPO counterpart or with the homologous milk-derived EPO-Fc. The role of the molecular weight seemed to be important in enhancing the hematopoietic activity of SiHa-derived EPO-Fc. However, the lack of sialylated multi-antennary glycosylation profile in milk-derived EPO-Fc resulted in lower biological activity. Conclusions The low content of tri- or tetra-antennary sialylated N-glycans linked to the chimeric EPO-Fc hormone, expressed in the goat mammary gland epithelial cells, defined its in vivo hematopoietic activity. General significance The sialylated N-glycan content plays a more significant role in the in vivo biological activity of hEPO than its increased molecular weight. [ABSTRACT FROM AUTHOR]