A novel dual-plasmid platform provides scalable transfection yielding improved productivity and packaging across multiple AAV serotypes and genomes.

Transient transfection of mammalian cells using plasmid DNA is a standard method to produce adeno-associated virus (AAV) vectors allowing for flexible and scalable manufacture. Typically, three plasmids are used to encode the necessary components to facilitate vector production; however, a dual-plasmid system, termed pDG, was introduced over 2 decades ago demonstrating two components could be combined resulting in comparable productivity to triple transfection. We have developed a novel dual-plasmid system, pOXB, with an alternative arrangement of sequences that results in significantly increased AAV vector productivity and percentage of full capsids packaged in comparison to the pDG dual design and triple transfection. Here, we demonstrate the reproducibility of these findings across seven recombinant AAV genomes and multiple capsid serotypes as well as the scalability of the pOXB dual-plasmid transfection at 50-L bioreactor scale. Purified drug substance showed a consistent product quality profile in line with triple-transfected vectors, except for a substantial improvement in intact genomes packaged using the pOXB dual- transfection system. Furthermore, pOXB dual- and triple-transfection-based vectors performed consistently in vivo. The pOXB dual plasmid represents an innovation in AAV manufacturing resulting in significant process gains while maintaining the flexibility of a transient transfection platform.

Widening the pH activity profile of a fungal laccase by directed evolution.

Unnatural selection: A fungal laccase was tailored by directed evolution to be active at neutral/alkaline pH. After five generations, the final mutant showed a broader pH profile while retaining 50 to 80 % of its activity at neutral pH.

Purification of Escherichia coli RNA polymerase using a self-cleaving elastin-like polypeptide tag.

A self-cleaving elastin-like polypeptide (ELP) tag was used to purify the multisubunit Escherichia coli RNA polymerase (RNAP) via a simple, nonchromatographic method. To accomplish this, the RNAP alpha subunit was tagged with a self-cleaving ELP-intein tag and coexpressed with the beta, beta', and omega subunits. The assembled RNAP was purified with its associated subunits, and was active and acquired at reasonable yield and purity. To remove residual polynucleotides bound to the purified RNAP, two polymer precipitation methods were investigated: polyethyleneimine (PEI) and polyethylene (PEG) precipitation. The PEG procedure was shown to enhance purity and was compatible with downstream ELP-intein purification. Thus, this simple ELP-based method should be applicable for the nonchromatographic purification of other recombinant, in vivo-assembled multisubunit complexes in a single step. Further, the simplicity and low cost of this method will likely facilitate scale up for large-scale production of additional multimeric protein targets. Finally, this technique may have utility in isolating protein interaction partners that associate with a given target.

Catalytic role of conserved HQGE motif in the CE6 carbohydrate esterase family.

An acetylxylan esterase (R.44), belonging to the carbohydrate esterase family 6 (CE6), retrieved from bovine rumen metagenome was analyzed. Molecular modelling and site-directed mutagenesis indicated that the enzyme possesses a catalytic triad formed by Ser(14), His(231) and Glu(152). The catalytic Ser and His have been identified in highly conserved sequences GQSX and DXXH in the CE6 family, respectively, and the active-site glutamate was part of a highly conserved sequence HQGE. This motif is situated near to the so-called Block III in the CE6 family and its role in catalysis has not been identified so far.

Biochemical and structural features of a novel cyclodextrinase from cow rumen metagenome.

A novel enzyme, RA.04, belonging to the alpha-amylase family was obtained after expression of metagenomic DNA from rumen fluid (Ferrer et al.: Environ. Microbiol. 2005, 7, 1996-2010). The purified RA.04 has a tetrameric structure (280 kDa) and exhibited maximum activity (5000 U/mg protein) at 70 degrees C and was active within an unusually broad pH range from 5.5 to 9.0. It maintained 80% activity at pH 5.0 and 9.5 and 75 degrees C. The enzyme hydrolyzed alpha-D-(1,4) bonds 13-fold faster than alpha-D-(1,6) bonds to yield maltose and glucose as the main products, and it exhibited transglycosylation activity. Its preferred substrates, in the descending order, were maltooligosaccharides (C3-C7), cyclomaltoheptaose (beta-CD), cyclomaltohexaose (alpha-CD), cyclomaltooctaose (gamma-CD), soluble starch, amylose, pullulan and amylopectin. The biochemical properties and amino acid sequence alignments suggested that this enzyme is a cyclomaltodextrinase. However, despite the similarity in the catalytic module (with Glu359 and Asp331 being the catalytic nucleophile and substrate-binding residues, respectively), the enzyme bears a shorter N-terminal domain that may keep the active site more accessible for both starch and pullulan, compared to the other known CDases. Moreover, RA.04 lacks the well-conserved N-terminal Trp responsible for the substrate preference typical of CDases/MAases/PNases, suggesting a new residue is implicated in the preference for cyclic maltooligosaccharides. This study has demonstrated the usefulness of a metagenomic approach to gain novel debranching enzymes, important for the bread/food industries, from microbial environments with a high rate of plant polymer turnover, exemplified by the cow rumen.

Purification and kinetic characterization of a fructosyltransferase from Aspergillus aculeatus.

A fructosyltransferase present in Pectinex Ultra SP-L, a commercial enzyme preparation from Aspergillus aculeatus, was purified to 107-fold and further characterised. The enzyme was a dimeric glycoprotein (20% (w/w) carbohydrate content) with a molecular mass of around 135 kDa for the dimer. Optimal activity/stability was found in the pH range 5.0-7.0 and at 60 degrees C. It was stable or slightly activated (upto 1.4-fold) in the presence of reducing agents, such as dithiothreitol and 2-mercaptoethanol, and detergents, such as sodium dodecylsulphate and Tween 80. The enzyme was able to transfer fructosyl groups from sucrose as donor producing the corresponding series of fructooligosaccharides: 1-kestose, nystose and fructosylnystose. Using sucrose as substrate, the k(cat) and K(m) values for transfructosylating activity were 1.62+/-0.09 x 10(4)s(-1) and 0.53+/-0.05 M, whereas for hydrolytic activity the corresponding values were 775+/-25s(-1) and 27+/-3 mM. At elevated sucrose concentrations, the fructosyltransferase from A. aculeatus showed a high transferase/hydrolase ratio that confers it a great potential for the industrial production of prebiotic fructooligosaccharides.

Beet sugar syrup and molasses as low-cost feedstock for the enzymatic production of fructo-oligosaccharides.

Sugar syrup and molasses from beet processing containing 620 and 570 mg/mL sucrose, respectively, were assayed as low-cost and available substrates for the enzymatic synthesis of fructo-oligosaccharides (FOSs). A commercial pectinase (Pectinex Ultra SP-L, from Aspergillus aculeatus) characterized by the presence of a transfructosylating activity was used as a biocatalyst. The FOS production increased when lowering the initial pH value of syrup (7.5) and molasses (8.9) to 5.5. Sugar syrup and molasses were diluted in order to reduce substrate viscosity; interestingly, the percentage of FOS with regards to total sugars remained almost constant, which indicated a high transferase-to-hydrolase ratio for this enzyme. Kinetics of FOS production was analyzed. Using approximately 10 U transfructosylating activity per g sucrose, the FOS concentration reached a maximum of 388 mg/mL after 30 h using syrup and 235 mg/mL in 65 h with molasses. These values corresponded to approximately 56 and 49% (w/w), respectively, of the total amount of carbohydrates in the mixture. The enzyme was also covalently immobilized on an epoxy-activated polymethacrylate-based polymer (Sepabeads EC-EP5). We found that immobilized Pectinex Ultra SP-L can be efficiently applied to the synthesis of FOS using syrup and molasses as substrates.