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Cellular factors which facilitate protein folding and disulphide bond formation: novel machinery for improved protein production by cell factories

Objective



This proposal is from a consortium of 8 groups in 6 EU member states. The aims are to improve understanding of the folding of secreted proteins and of the cellular factors which assist folding, and to exploit this to improve the efficiency of expression of correctly-folded high value recombinant proteins. The proposal addresses key issues highlighted in Cell Factories (Area 1) of the Work Programme in Biotechnology 1994-1998.
There will be significant opportunities for European industry to exploit outputs from the programme, as indicated by the participation of Zeneca and the strong expressions of support from other major companies.
The production of high value proteins in convenient host systems by use of recombinant DNA technology, is a major activity of the biotechnology industry. The markets for such proteins are in human/veterinary medicine, as pharmaceuticals, diagnostics or vaccines, and in the food or fine chemical industries as industrial enzymes. The great majority of the proteins of interest are extracellular proteins which, in the course of biosynthesis and secretion, undergo a complex series of steps involving targeting, folding, modification and assembly.
It is a considerable challenge to reproduce this series of events faithfully in a high-yield recombinant protein production system; many systems yield products which are misfolded (aggregated and lacking biological activity). In particular, secreted proteins are stabilised by intra- and inter-molecular disulphide bonds; formation of the correct disulphide bonds requires oxidising conditions and an appropriate set of enzymes to catalyse disulphide formation and isomerization.
The aim of the proposed programme is to extend knowledge of these enzymes in bacteria and eukaryotic cells and to apply them to manipulate host cell expression systems so that high levels of desired recombinant proteins can be combined with efficient correct folding.
The programme will fall into 3 sections: A) studies on the pathways of folding of challenging disulphide-bonded model proteins, based on recent advances in mass spectrometry, B) studies on the catalysts of protein disulphide bond formation in eukaryotes and prokaryotes, and C) studies in-vivo on cell and medium engineering to optimise expression of proteins. Information will flow from A to B to C, permitting exploitation in vívo of understanding of the properties of the catalysts in vi tro .
The consortium comprises world-leading laboratories in the study of the key enzymes, in the molecular analysis of protein folding pathways and in industrial-scale protein folding. Each partner in the programme will interact with several others and the whole programme will operate as a network. The programme is totally dependent on this trans-national collaboration and could not be conducted without support at this level; the collaborators bring different skills, facilities and background experience to the programme and form a very strong European team.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

UNIVERSITY OF KENT AT CANTERBURY
Address

CT2 7NZ Folkestone
United Kingdom

Participants (7)

Commissariat a l'Energie Atomique
France
Address
Ctr Etudes De Saclay - Ce-s
91191 Gif-sur-yvette
KAROLINSKA INSTITUTE
Sweden
Address
9,Doktorsringen 9 A
171 77 Stockholm
Martin-Luther-Universitat Halle-Wittenberg
Germany
Address
16 A,weinbergweg 16 A
6120 Halle
UNIVERSITA DEGLI STUDI DI NAPOLI FEDERICO II
Italy
Address
Via Mezzocannone 16
80134 Napoli
UNIVERSITAET REGENSBURG
Germany
Address
Universitaetsstrasse 31
93040 Regensburg
University of Oulu
Finland
Address
52 A,kajaanintie 52 A
90220 Oulu
ZENECA LTD.
United Kingdom
Address
Stanhope Gate 15
W1Y 6LN London