Ziel
To exchange and improve the knowledge with regard to the quality and quantity of heterologous protein expression in bacteria with a special emphasis on antibodies.
A workshop has been held with all participants as well as a group of invited experts in the field entitled 'Genetic technologies for Developing Cell Factories' It took place from 22-24 June, 1995 in Trieste, Italy.
- The basis for a new type selection system based on antibody antigen interactions has been developed.
- The SRP pathway has been resolved in Bacillus.
- A concept has been developed for the direct selection of bispecific antibody fragments from large phage display libraries
- It was shown that expression of antibody fragments in E. coli can be dramatically improved by replacing critical amino acids in the framework.
MAJOR SCIENTIFIC BREAKTHROUGHS:
- The filamentous phages fd and IKe infect their host by means of pili on the host membrane. fd infect bacteria bearing F pile, whereas IKe infects bacteria bearing N or I pili. Infection is mediated by the gene 3 protein (g3p). The host range of fd phage was augmented by grafting different parts of IKe g3p to the end of fd g3p. Phage bearring such chimeric g3p are able to infect bacteria bearing both N and F pili provinding they contain at least the receptor domain IKe g3p. By adding the glycine rich domain high levels of infection were obtained. This could provide the basis for a selection system based on antibody antigen interaction if the N pilus protein recognised by the IKe g3p is shown to be permissive to the insertion of foreign epitopes.
- The SRP pathway has been resolved in Bacillus. The ftsY gene (docking protein homologue) was characterised. The Bacillus docking protein appears to be smaller than the eukaryotic counterpart. It lacks a clear membrane spanning domain. FtsY is located on the chromosome in close proximity to the FfH protein (54 homologue) another essential building block of the signal recognition particle. This is an indication for the concerted function of these two genes. The ftsY gene is essential for Bacillus as concluded from deletion experiments. Its expression is, interestingly, highest during logarithmic phase and not in stationary phase where protein secretion is highest.
- The potential of diabodies to provide a more versatile format for the development of bispecific antibody fragments was explored. Repertoires of V-genes from immunised mice were assembled in the diabody format an cloned in phage display vectors. This technology lends itself to the construction of very large numbers of diabody clones which can be selected on two different antigens to enrich for bispecific binders. As a model system 2-phenyloxazol-5-one and digoxigenin were used as antigens. It was demonstrated that large numenrs of mono- and bispecific diabodies can be cloned in a stable format and expressed on the surface of phage.
- Aggregation is one of the most imported limiting factors in producing heterologous proteins in E. coli. Using antibody fragments is was found that sequence factors are responsible for this behaviour. From a detailed sequence comparison of only framework residues, using single and double mutations, it was possible to narrow the effect to only three residues, which act synergistically. The mutations affect the aggregation reaction itself. Using such improved variants it was possible to obtain yields as high as 3-4 gram per liter in high cell density fermentation.
- A tightly controllable expression system for the simultaneous and stringent regulation of several genes in E. coli was developed. The system allows not only to create 'on/off' situations of gene activities but also to adjust intracellular concentrations of gene products with high precision. The system is based on three families of components. 1) A set of promoters which can tightly regulated, controlled by the repressor/operator elements of the lac or tetracycline resistance operon (lacR/O; tetR/O) and the transcription activator AraC. 2) A family of plasmids of congruent molecular structure. 3) E. coli strains which contain chromosomally located expression units producing constitutively proper amounts of tet repressor (tetR), lac repressor (lacR) and AraC.
The potential of this regulatory system has been demonstrated by cloning and expressing a nuclease gene.
- Highly specific antibodies directed against the midgut of lepidopteran insects were fused to Bacillus thuringiensis delta-endotoxins to be used as 'immunotoxins' with improved specificity. In addition single-chain antibodies directed against viral plant pathogens were fused with alkaline phosphatase (AP) for applications in detection. An important advantage of the latter is that AP is a dimer resulting in dimeric scFvs.
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6700 ES Wageningen
Niederlande