To interact with their environment, cells depend on secreted and membrane proteins. In eukaryotic cells, these proteins are produced in the endoplasmic reticulum (ER), an organelle dedicated to protein biogenesis. An understanding of how proteins acquire their native structure in the ER and how cells control and regulate this process is a prerequisite for targeted interventions in molecular engineering and human therapy. Using state-of-the-art approaches, we provide fundamental insights into the inner workings of cells and use these insights for rational molecular engineering in the following areas: