During heterologous protein production with Escherichia coli, the formation of inclusion bodies (IBs) is often a major drawback as these aggregated proteins are usually inactive. However, different strategies for the generation of IBs consisting of catalytically active proteins have recently been described. In this study, the archaeal tetrameric coiled-coil domain of the cell-surface protein tetrabrachion was fused to a target reporter protein to produce fluorescent IBs (FIBs). As the cultivation conditions severely influence IB formation, the entire cultivation process resulting in the production of FIBs were thoroughly studied. First, the cultivation process was scaled down based on the maximum oxygen transfer capacity, combining online monitoring technologies for shake flasks and microtiter plates with offline sampling. The evaluation of culture conditions in complex terrific broth autoinduction medium showed strong oxygen limitation and leaky expression. Furthermore, strong acetate formation and pH changes from 6.5 to 8.8 led to sub-optimal cultivation conditions. However, in minimal Wilms-MOPS autoinduction medium, defined culture conditions and a tightly controlled expression were achieved. The production of FIBs is strongly influenced by the induction strength. Increasing induction strengths result in lower total amounts of functional protein. However, the amount of functional FIBs increases. Furthermore, to prevent the formation of conventional inactive IBs, a temperature shift from 37 °C to 15 °C is crucial to generate FIBs. Finally, the gained insights were transferred to a stirred tank reactor batch fermentation. Hereby, 12 g/L FIBs were produced, making up 43 % (w/w) of the total generated biomass.