production of recombinant rankl for in vitro osteoclastogenesis
Recombinant expression of antibody fragments in E. coli is often hampered by low expression, inefficient translocation and aggregation in the periplasm. Aggregation problem during in vitro-renaturation is also a limiting factor, when antibody fragments expressed in bacterial cytosol as inclusion bodies. The objective of this work was to achieve and optimise periplasmic production, and to explore novel strategies for in vitro renaturation from cytoplasmic IB’s. For the study, the single chain antibody fragment against the hapten oxazolone was chosen as the model system. In this book, results on periplasmic expression and expression as inclusion bodies and refolding thereof are presented. In expression in periplasmic effect of promoter strength and growth cultivation are presented. In vitro renaturation of IB's lead to establishment refolding protocols comprising of Ionic Liquids, which are emerging as green alternatives. Besides, we demonstrated usage of aromatic thiols as redox buffers components for protein refolding. Findings presented herein might be helpful for the of the periplasmic production of antibody fragments as as well as for establish protocols at physiological pH.