Combined clarification and affinity capture using magnetic resin enables efficient separation of rAAV5 from cell lysate.

Recombinant adeno-associated virus (rAAV) vectors have displayed enormous potential as a platform for delivery of gene therapies. Purification of rAAV at industrial scale involves a series of elaborate, material, and time-consuming midstream steps, such as clarification by depth filtration and concentration/buffer exchange by tangential flow filtration. In this study, we developed a filter-less flow capture method for purification of rAAV serotype 5, using a high-gradient magnetic separator and magnetic Mag Sepharose beads coupled to an AVB affinity ligand. In under 2 h, we captured and eluted rAAV5 directly from ∼5 L of cell lysate with a recovery yield of 63% (±5%, n = 3). Compared to cell lysate, the eluate showed a 3-log reduction of host cell DNA and host cell proteins. The process developed eliminates the need for filtration and column chromatography in the early steps of industrial rAAV purification. This will be of high value for industrial-scale manufacturing of rAAVs by reducing time and material in the purification process, without compromising product recovery and purity.

Generation of a functional, semisynthetic [FeFe]-hydrogenase in a photosynthetic microorganism.

[FeFe]-Hydrogenases are hydrogen producing metalloenzymes with excellent catalytic capacities, highly relevant in the context of a future hydrogen economy. Here we demonstrate the synthetic activation of a heterologously expressed [FeFe]-hydrogenase in living cells of Synechocystis PCC 6803, a photoautotrophic microbial chassis with high potential for biotechnological energy applications. H2-Evolution assays clearly show that the non-native, semi-synthetic enzyme links to the native metabolism in living cells.

Design and characterization of a synthetic minimal promoter for heterocyst-specific expression in filamentous cyanobacteria.

Short and well defined promoters are essential for advancing cyanobacterial biotechnology. The heterocyst of Nostoc sp. is suggested as a microbial cell factory for oxygen sensitive catalysts, such as hydrogenases for hydrogen production, due to its microoxic environment. We identified and predicted promoter elements of possible significance through a consensus strategy using a pool of heterocyst-induced DIF+ promoters known from Anabaena sp. PCC 7120. To test if these conserved promoter elements were crucial for heterocyst-specific expression, promoter-yfp reporter constructs were designed. The characterization was accomplished by replacing, -35 and -10 regions and the upstream element, with well described elements from the trc promoter of Escherichia coli, which is also functional in Nostoc sp. From the in vivo spatial fluorescence of the different promoter-yfp reporters in Nostoc punctiforme ATCC 29133, we concluded that both the consensus -35 and extended -10 regions were important for heterocyst-specific expression. Further that the promoter strength could be improved by the addition of an upstream element. We designed a short synthetic promoter of 48 nucleotides, PsynDIF, including a consensus DIF1 sequence, a 17 base pair stretch of random nucleotides and an extended consensus -10 region, and thus generated the shortest promoter for heterocyst-specific expression to date.