An Air-Well sparging minifermenter system for high-throughput protein production.

BACKGROUND: Over the last few years High-Throughput Protein Production (HTPP) has played a crucial role for functional proteomics. High-quality, high yield and fast recombinant protein production are critical for new HTPP technologies. Escherichia coli is usually the expression system of choice in protein production thanks to its fast growth, ease of handling and high yields of protein produced. Even though shake-flask cultures are widely used, there is an increasing need for easy to handle, lab scale, high throughput systems. RESULTS: In this article we described a novel minifermenter system suitable for HTPP. The Air-Well minifermenter system is made by a homogeneous air sparging device that includes an air diffusion system, and a stainless steel 96 needle plate integrated with a 96 deep well plate where cultures take place. This system provides aeration to achieve higher optical density growth compared to classical shaking growth without the decrease in pH value and bacterial viability. Moreover the yield of recombinant protein is up to 3-fold higher with a considerable improvement in the amount of full length proteins. CONCLUSIONS: High throughput production of hundreds of proteins in parallel can be obtained sparging air in a continuous and controlled manner. The system used is modular and can be easily modified and scaled up to meet the demands for HTPP.

Coating of nitrocellulose for colorimetric DNA microarrays.

We report on the modification of a nitrocellulose film with copoly(DMA-NAS-MAPS), a tercopolymer based on N,N-dimethylacrylamide (DMA), N-acryloyloxysuccinimide (NAS), and 3-(trimethoxysilyl)propyl-methacrylate (MAPS). The chains of this polymer, interacting with nitrocellulose fibers, introduce active ester functionalities that promote the covalent binding of short oligonucleotide fragments to the nitrocellulose thin film. Using colorimetric detection, naked eye visible DNA microarrays are developed for easy identification of foodborne pathogens. The fast and robust procedure of nitrocellulose functionalization opens the opportunity to implement this material in disposable analytical microdevices that do not require sophisticated readout systems.

Epitope mapping of human chromogranin A by peptide microarrays.

In this chapter we report on the characterization of linear antigenic sites of human chromogranin A (CgA), a useful tissue and serum marker for neuroendocrine tumours and a precursor of many biologically active peptides. The epitope mapping of CgA has been carried out by peptide microarrays on glass slides coated by a copolymer of N,N-dimethylacrylamide (DMA), N,N-acryloyloxysuccinimide (NAS) and [3-(methacryloyl-oxy) propyl] trimethoxysilyl (MAPS). The microarray support provided sufficient accessibility of the ligand, with no need for a spacer, as the polymer chains prevent interaction of immobilized peptides with substrate. In addition, the polymeric surface constitutes an aqueous micro-environment in which, despite peptide random orientation, linear epitopes are freely exposed. The results reported are in accordance with those obtained in conventional ELISA assays using biotinylated and non-biotinylated peptides.