Engineering Saccharomyces cerevisiae for the production and secretion of Affibody molecules.

BACKGROUND: Affibody molecules are synthetic peptides with a variety of therapeutic and diagnostic applications. To date, Affibody molecules have mainly been produced by the bacterial production host Escherichia coli. There is an interest in exploring alternative production hosts to identify potential improvements in terms of yield, ease of production and purification advantages. In this study, we evaluated the feasibility of Saccharomyces cerevisiae as a production chassis for this group of proteins. RESULTS: We examined the production of three different Affibody molecules in S. cerevisiae and found that these Affibody molecules were partially degraded. An albumin-binding domain, which may be attached to the Affibody molecules to increase their half-life, was identified to be a substrate for several S. cerevisiae proteases. We tested the removal of three vacuolar proteases, proteinase A, proteinase B and carboxypeptidase Y. Removal of one of these, proteinase A, resulted in intact secretion of one of the targeted Affibody molecules. Removal of either or both of the two additional proteases, carboxypeptidase Y and proteinase B, resulted in intact secretion of the two remaining Affibody molecules. The produced Affibody molecules were verified to bind their target, human HER3, as potently as the corresponding molecules produced in E. coli in an in vitro surface-plasmon resonance binding assay. Finally, we performed a fed-batch fermentation with one of the engineered protease-deficient S. cerevisiae strains and achieved a protein titer of 530 mg Affibody molecule/L. CONCLUSION: This study shows that engineered S. cerevisiae has a great potential as a production host for recombinant Affibody molecules, reaching a high titer, and for proteins where endotoxin removal could be challenging, the use of S. cerevisiae obviates the need for endotoxin removal from protein produced in E. coli.

High throughput generation of a resource of the human secretome in mammalian cells.

The proteins secreted by human tissues and blood cells, the secretome, are important both for the basic understanding of human biology and for identification of potential targets for future diagnosis and therapy. Here, a high-throughput mammalian cell factory is presented that was established to create a resource of recombinant full-length proteins covering the majority of those annotated as 'secreted' in humans. The full-length DNA sequences of each of the predicted secreted proteins were generated by gene synthesis, the constructs were transfected into Chinese hamster ovary (CHO) cells and the recombinant proteins were produced, purified and analyzed. Almost 1,300 proteins were successfully generated and proteins predicted to be secreted into the blood were produced with a success rate of 65%, while the success rates for the other categories of secreted proteins were somewhat lower giving an overall one-pass success rate of ca. 58%. The proteins were used to generate targeted proteomics assays and several of the proteins were shown to be active in a phenotypic assay involving pancreatic ß-cell dedifferentiation. Many of the proteins that failed during production in CHO cells could be rescued in human embryonic kidney (HEK 293) cells suggesting that a cell factory of human origin can be an attractive alternative for production in mammalian cells. In conclusion, a high-throughput protein production and purification system has been successfully established to create a unique resource of the human secretome.

The human secretome.

The proteins secreted by human cells (collectively referred to as the secretome) are important not only for the basic understanding of human biology but also for the identification of potential targets for future diagnostics and therapies. Here, we present a comprehensive analysis of proteins predicted to be secreted in human cells, which provides information about their final localization in the human body, including the proteins actively secreted to peripheral blood. The analysis suggests that a large number of the proteins of the secretome are not secreted out of the cell, but instead are retained intracellularly, whereas another large group of proteins were identified that are predicted to be retained locally at the tissue of expression and not secreted into the blood. Proteins detected in the human blood by mass spectrometry-based proteomics and antibody-based immunoassays are also presented with estimates of their concentrations in the blood. The results are presented in an updated version 19 of the Human Protein Atlas in which each gene encoding a secretome protein is annotated to provide an open-access knowledge resource of the human secretome, including body-wide expression data, spatial localization data down to the single-cell and subcellular levels, and data about the presence of proteins that are detectable in the blood.

Order, Disorder, and Temperature-Driven Compaction in a Designed Elastin Protein.

Artificial minielastin constructs have been designed that replicate the structure and function of natural elastins in a simpler context, allowing the NMR observation of structure and dynamics of elastin-like proteins with complete residue-specific resolution. We find that the alanine-rich cross-linking domains of elastin have a partially helical structure, but only when capped by proline-rich hydrophobic domains. We also find that the hydrophobic domains, composed of prominent 6-residue repeats VPGVGG and APGVGV found in natural elastins, appear random coil by both NMR chemical shift analysis and circular dichroism. However, these elastin hydrophobic domains exhibit structural bias for a dynamically disordered conformation that is neither helical nor ß sheet with a degree of nonrandom structural bias which is dependent on residue type and position in the sequence. Another nonrandom-coil aspect of hydrophobic domain structure lies in the fact that, in contrast to other intrinsically disordered proteins, these hydrophobic domains retain a relatively condensed conformation whether attached to cross-linking domains or not. Importantly, these domains and the proteins containing them constrict with increasing temperature by up to 30% in volume without becoming more ordered. This property is often observed in nonbiological polymers and suggests that temperature-driven constriction is a new type of protein structural change that is linked to elastin's biological functions of coacervation-driven assembly and elastic recoil.