The Protein Factory is a new Center currently being established at the Politecnico of Milano and University of Insubria in Varese to promote basic and applied research on proteins of biotechnological relevance
The Center will comprise a wide range of expertise and skills within its research units, with activities in the areas of protein engineering, proteomics, high throughput protein production, chemical biology, biocatalysis, green chemistry, cell biology, and protein therapeutics. The Centre will also contribute to the progress of translational research within industry and medicine and provide fundamental insights which can be used to promote protein discovery and development.
The aim of “The Protein Factory” is to offer an integrated laboratory for the production and characterization of recombinant proteins useful in the different fields of biotechnology. In order to reach the critical size required to manage complex projects in a strategic sector of the biomedical and industrial research, this Center will join the expertises and skills of the two proponent institutions.
We apply a flexible approach to each single project to significantly reduce the time and ultimately the cost associated with protein production. Years of solid experience and unique expertise in protein expression and purification allows to this protein core facility to break the bottlenecks in protein expression and purification.
· Gene design based on bioinformatics analysis and codon optimization technology.
· PCR cloning and subcloning.
· Plasmid preparation and protein expression in bacteria, yeast and eukaryotic cells. Our cloning technology enables cloning of a gene into a wide range of vectors both commercial and custom made and allows:
- expression from different promoters or native expression
- inclusion of cleavable affinity tags
- cytosolic or periplasmic localisation.
· Rapid identification of optimum combination of vector and expression conditions.
· Purification, including ion exchange, affinity, gel filtration and hydrophobic column.
· Scale-up of recombinant proteins with a production capacity of up to 25 litres.
Recent developments in directed evolution technologies combined with innovations in computational and screening methods have revolutionized protein engineering. More specifically, rational design (based on structural analysis and site-directed mutagenesis) and directed evolution (based on combinatorial methods such as DNA shuffling and other methods of genetic recombination) have resulted in the improvement of proteins of industrial or therapeutic interest. At “The Protein Factory” we developed a specific “combined approach” to produce in a comparatively short period of time new enzymatic activities for defined applications starting from a common scaffold. The proteins are finalized to new developments in enzymatic catalysis (Biocatalysis)
Following the expression of recombinant proteins, the expertise and instrumentations available at “The Protein Factory” allows a plethora of functional and structural characterization studies:
- set up of specific assay methods
- folding studies
- UV-visible, fluorescence and CD spectroscopy
- kinetic characterization.
- Enzyme reaction mechanisms
Applications take advantage from the novel powerfull technique for bringing to the limelight the “hidden proteome” from natural source, based on a capture on hexa-peptide baits.
Directed evolution is a term used to describe various techniques for generation of protein mutants and selection of desirable functions. In the past years, directed protein evolution has emerged as a powerful way in protein engineering, in particular of enzyme activities. This technology has been advanced considerably by the availability of molecular biology tools and emerging high-throughput screening technologies. Directed protein evolution allows to gradually accumulate mutations, either sequentially or by recombination, while applying selective pressure. This is achieved by the generation of libraries of mutants followed by the screening for targeted functions and subsequent repetition of the process using improved mutants from the previous screening.
Advanced recombinant DNA technologies have then allowed the transfer of genes to a suitable host for rapid propagation and/or high level protein production. The development of small size and automatizable chemical or biological assays allow the screening of large numbers of samples for selection of desired functions.
The availability of protein variants provides insight into its sequence-function relationships and allows to select the strategy best suited for the evolution of a specific protein.
“We have just begun to see the impacts of protein engineering on biotechnology”