Refolding of Npro fusion proteins.

The autoprotease Npro significantly enhances expression of fused peptides and proteins and drives the formation of inclusion bodies during protein expression. Upon refolding, the autoprotease becomes active and cleaves itself specifically at its own C-terminus releasing the target protein with its authentic N-terminus. Npro wild-type and its mutant EDDIE, respectively, were fused N-terminally to the model proteins green fluorescent protein, staphylococcus Protein A domain D, inhibitory peptide of senescence-evasion-factor, and the short 16 amino acid peptide pep6His.

N(pro) fusion technology to produce proteins with authentic N termini in E. coli.

We describe a prokaryotic expression system using the autoproteolytic function of N(pro) from classical swine fever virus. Proteins or peptides expressed as N(pro) fusions are deposited as inclusion bodies. On in vitro refolding by switching from chaotropic to kosmotropic conditions, the fusion partner is released from the C-terminal end of the autoprotease by self-cleavage, leaving the target protein with an authentic N terminus.

Improvement of bioprocess monitoring: development of novel concepts.

The advancement of bioprocess monitoring will play a crucial role to meet the future requirements of bioprocess technology. Major issues are the acceleration of process development to reduce the time to the market and to ensure optimal exploitation of the cell factory and further to cope with the requirements of the Process Analytical Technology initiative. Due to the enormous complexity of cellular systems and lack of appropriate sensor systems microbial production processes are still poorly understood.

Sensor combination and chemometric modelling for improved process monitoring in recombinant E. coli fed-batch cultivations.

The key objective for the optimisation of recombinant protein production in bacteria is to optimize the exploitation of the host cell's synthesis potential. Recent studies show that the novel concept of transcription rate control allows the tuning of recombinant gene expression in relation to the metabolic capacity of the host cell. To adjust the inducer-biomass ratio to a tolerable level, real-time knowledge about key process variables is paramount.

Monitoring of protein profiles for the optimization of recombinant fermentation processes using public domain databases.

The expression of human superoxide dismutase in fed-batch fermentation of E. coli HMS174(DE3)(pET3ahSOD) was studied as model system. Due to the frequently used strong T7 promoter system a high metabolic load is exerted, which triggers stress response mechanisms and finally leads to the differentiation of the host cell.