Expression and downstream purification of insulin molecules in Pichia pastoris

In the next decade and beyond global demand for insulin is expected to rise significantly requiring additional manufacturing capacity. The next generation of insulin manufacturing plants will likely be based on new and robust expression and bioprocess platforms that are flexible, safe, simple to implement in a manufacturing setting and capable of step improvements in productivity and cost compared to current manufacturing techniques. Towards this end, Pichia pastoris expression systems has been evaluated for insulin due to its capacity to secrete a variety of heterologous proteins and its ability to grow to high cell densities. Under the well-characterized, tightly regulated AOX1 promoter, yields of 1.5 to nearly 3 g/L of purified insulin have been reported.1,2,3 However, methanol is a very volatile substance requiring specialized facilities, which can hamper large-scale production. Downstream processing of insulin precursors also requires use of organic solvents which can also burden manufacturing. We report development of an insulin process using a constitutive promoter expression system in place of the inducible AOX1 promoter, and a simplified downstream purification process using precipitation. Fermentations were carried out in 2 L scale bioreactors and culture supernatant collected after 65 hours. A design of experiment (DoE) was performed to identify optimal conditions for polyelectrolyte precipitation of the recombinant protein using polyvinyl sulfonic acid (PVS).4 The resulting pellets were then analyzed via SDS-PAGE and HPLC. 1. Gurramkonda, C. et al. Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin. Microb. Cell Fact. 9, 31 (2010). 2. Polez, S. et al. A Simplified and Efficient Process for Insulin Production in Pichia pastoris. PLoS One 11, e0167207 (2016). 3. Xie, T., Liu, Q., Xie, F., Liu, H. & Zhang, Y. Secretory expression of insulin precursor in pichia pastoris and simple procedure for producing recombinant human insulin. Prep. Biochem. Biotechnol. 38, 308–17 (2008). 4. Buddha, M. et al. Precipitation as an Alternative to Chromatography in the Insulin Manufacturing Process. BioPharm Int. 30–36 (2016)

The multifunctional autophagy pathway in the human malaria parasite, Plasmodium falciparum.

Autophagy is a catabolic pathway typically induced by nutrient starvation to recycle amino acids, but can also function in removing damaged organelles. In addition, this pathway plays a key role in eukaryotic development. To date, not much is known about the role of autophagy in apicomplexan parasites and more specifically in the human malaria parasite Plasmodium falciparum. Comparative genomic analysis has uncovered some, but not all, orthologs of autophagy-related (ATG) genes in the malaria parasite genome. Here, using a genome-wide in silico analysis, we confirmed that ATG genes whose products are required for vesicle expansion and completion are present, while genes involved in induction of autophagy and cargo packaging are mostly absent. We subsequently focused on the molecular and cellular function of P. falciparum ATG8 (PfATG8), an autophagosome membrane marker and key component of the autophagy pathway, throughout the parasite asexual and sexual erythrocytic stages. In this context, we showed that PfATG8 has a distinct and atypical role in parasite development. PfATG8 localized in the apicoplast and in vesicles throughout the cytosol during parasite development. Immunofluorescence assays of PfATG8 in apicoplast-minus parasites suggest that PfATG8 is involved in apicoplast biogenesis. Furthermore, treatment of parasite cultures with bafilomycin A 1 and chloroquine, both lysosomotropic agents that inhibit autophagosome and lysosome fusion, resulted in dramatic morphological changes of the apicoplast, and parasite death. Furthermore, deep proteomic analysis of components associated with PfATG8 indicated that it may possibly be involved in ribophagy and piecemeal microautophagy of the nucleus. Collectively, our data revealed the importance and specificity of the autophagy pathway in the malaria parasite and offer potential novel therapeutic strategies

Structures and Bioactivities of Dihydrochalcones from Metrodorea stipularis

Metrodorea stipularis stem extracts were studied in the search for possible antichagastic, antimalarial, and antitumoral compounds using cruzain from Trypanosoma cruzi, Plasmodium falciparum, and cathepsins B and L, as molecular targets, respectively. Dihydrochalcones 1, 2, 3, and 4 showed significant inhibitory activity against all the targets. Compounds 1-4 displayed IC50 values ranging from 7.7 to 21.6 mu M against cruzain; dihydrochalcones 2 and 4 inhibited the growth of three different strains of P. falciparum in low micromolar concentrations; and against cathepsins B and L these compounds presented good inhibitory activity with IC50 values ranging from 1.0 to 14.9 mu M. the dihydrochalcones showed good selectivity in their inhibitory activities against the cysteine proteases.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Fed Sao Carlos, Dept Quim, BR-13565905 Sao Carlos, SP, BrazilUniv Calif Riverside, Dept Cell Biol & Neurosci, Riverside, CA 92521 USAUniversidade Federal de São Paulo, BR-04039002 São Paulo, BrazilUniversidade Federal de São Paulo, BR-04039002 São Paulo, BrazilFAPESP: 2010/00496-8Web of Scienc

The multifunctional autophagy pathway in the human malaria parasite, <i>Plasmodium falciparum</i>

<p>Autophagy is a catabolic pathway typically induced by nutrient starvation to recycle amino acids, but can also function in removing damaged organelles. In addition, this pathway plays a key role in eukaryotic development. To date, not much is known about the role of autophagy in apicomplexan parasites and more specifically in the human malaria parasite <i>Plasmodium falciparum</i>. Comparative genomic analysis has uncovered some, but not all, orthologs of autophagy-related (<i>ATG</i>) genes in the malaria parasite genome. Here, using a genome-wide in silico analysis, we confirmed that <i>ATG</i> genes whose products are required for vesicle expansion and completion are present, while genes involved in induction of autophagy and cargo packaging are mostly absent. We subsequently focused on the molecular and cellular function of <i>P. falciparum</i> ATG8 (PfATG8), an autophagosome membrane marker and key component of the autophagy pathway, throughout the parasite asexual and sexual erythrocytic stages. In this context, we showed that PfATG8 has a distinct and atypical role in parasite development. PfATG8 localized in the apicoplast and in vesicles throughout the cytosol during parasite development. Immunofluorescence assays of PfATG8 in apicoplast-minus parasites suggest that PfATG8 is involved in apicoplast biogenesis. Furthermore, treatment of parasite cultures with bafilomycin A₁ and chloroquine, both lysosomotropic agents that inhibit autophagosome and lysosome fusion, resulted in dramatic morphological changes of the apicoplast, and parasite death. Furthermore, deep proteomic analysis of components associated with PfATG8 indicated that it may possibly be involved in ribophagy and piecemeal microautophagy of the nucleus. Collectively, our data revealed the importance and specificity of the autophagy pathway in the malaria parasite and offer potential novel therapeutic strategies.</p

Structures and Bioactivities of Dihydrochalcones from <i>Metrodorea stipularis</i>

<i>Metrodorea stipularis</i> stem extracts were studied in the search for possible antichagastic, antimalarial, and antitumoral compounds using cruzain from <i>Trypanosoma cruzi</i>, <i>Plasmodium falciparum</i>, and cathepsins B and L, as molecular targets, respectively. Dihydrochalcones <b>1</b>, <b>2</b>, <b>3</b>, and <b>4</b> showed significant inhibitory activity against all the targets. Compounds <b>1</b>–<b>4</b> displayed IC₅₀ values ranging from 7.7 to 21.6 μM against cruzain; dihydrochalcones <b>2</b> and <b>4</b> inhibited the growth of three different strains of <i>P. falciparum</i> in low micromolar concentrations; and against cathepsins B and L these compounds presented good inhibitory activity with IC₅₀ values ranging from 1.0 to 14.9 μM. The dihydrochalcones showed good selectivity in their inhibitory activities against the cysteine proteases