PCUBE

Infrastructure for Protein Production Platforms

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 Obiettivo del progetto (Objective)

'The most important prerequisite and challenge in structural biology research at the atomic level by any method is the availability of sufficiently large amounts of highly purified functional proteins due to the increasing size and complexity of target proteins or protein complexes to be analyzed. Heterologous expression in bacteria, yeast, insect, or mammalian cells combined with many attempts involving different DNA constructs that result in a large variety of protein variants is instrumental to obtain sufficiently high yields. This means high-throughput methods employing robotics and specialized infrastructure are essential to efficiently reach the state of obtaining functional protein and/or crystals for X-ray structure determination. PCUBE ('protein production platform') combines existing infrastructures and know-how of leading European laboratories in bacterial, eukaryotic expression of proteins, in high-throughput crystallization and in libraries design for the effective production and crystallization of macromolecules. The program aims at offering these various state-of-the-art platforms to research groups from EC member and associated states for the efficient production of proteins for their structural studies. Each infrastructure will offer defined procedures for applying for transnational access. Scientific selection committees will give priorities to applications by scientific criteria of excellence only. Researchers from the different sites offering infrastructure are combining efforts to improve the methods in the various areas by collaborating by joint research activities. These include the improvement of the automation for synthesising DNA constructs, for parallel expression, for improved libraries, for the efficient selection of affinity molecules from libraries to particular target proteins as well as developing new methods for crystallization at the nanoliter scale.'

Introduzione (Teaser)

Cutting-edge research in structural biology requires high-throughput and high-quality protein production platforms. European partners with requisite know-how joined forces to realise such platforms via transnational access (TNA).

Descrizione progetto (Article)

The EU-funded 'Infrastructure for protein production platforms' (http://www.p-cube.eu (PCUBE)) project worked on developing, improving and standardising protein production, screening and crystallisation technologies.

PCUBE members were highly successful in their endeavours. To realise high-throughput production, they developed robot-friendly protocols for automation. They also optimised and standardised molecular biology methods for protein selection and 3D characterisation as well as DNA purification to improve protein quality. Other areas of focus included optimising light microscopy techniques and establishing efficient protein expression systems in prokaryotic, insect and mammalian cells.

TNA was granted to 384 scientists and their projects from 30 different European countries. In parallel, 10 training events were offered on protein production, purification, expression, characterisation and crystallisation methods as well as protein engineering technologies.

Proteins' production in high quality and quantity is now possible through systems such as the MultiBac uses a baculovirus to reprogramme insect cell cultures and is harmless to humans. They optimised it to produce large numbers of desired multi-protein complexes.

Many European scientists accessed this patented technology through PCUBE TNA to produce proteins-of-interest. For instance, SweetBac was engineered that has untapped potential with regard to antibody production.

PCUBE funding helped realise the automation of a protein complex screening method that led to the development of the CoESPRIT technology. This was utilised by TNA users to further optimise high-throughput cloning. Automation for the efficient selection and production of designed ankyrin repeat proteins (DARPins) was also funded by PCUBE. Several binders were developed for specific protein targets using DARPin technology and distributed to TNA users for research.

The project also enabled the in vivo monitoring of live cells and ongoing protein interactions through techniques such as fluorescence cross-correlation spectroscopy and spectral position determination microscopy. Low-cost high-throughput technologies for protein crystallisation and in situ X-ray diffraction were developed that are robust and accurate.

PCUBE has successfully brought about technological innovation by setting benchmarks in structural biology and TNA to high precision instrumentation via joint research activities. Besides basic research, applications of PCUBE technologies include food security, environment protection, biomedicine, green energy and biosensing.