The picobodies are novel mini-proteins that were discovered recently in cattle. The picobodies combine several exciting features - small size, disulfide-rich and rigid structure, strong binding to pharmacological targets, and immense sequence diversity. Currently, the only way to discover new biologically active picobodies is to immunize cattle. The bovine immune system produces the picobodies as fusion proteins with a much larger antibody scaffold. Because of this, little is known about the folding, structure and activity of isolated picobodies. However, the isolated picobodies are exciting potential biotherapeutics that have a miniature and very stable structure that can bind to pharmacological targets with high affinity. In this project, we will investigate (i) the affinity maturation picobodies in vitro (i.e., to circumvent cattle immunization), (ii) test the production of isolated picobodies in yeast and bacteria, and (iii) systematically study the structure, folding and activity of isolated picobodies in comparison to their natural counterparts (picobodies fused to a larger antibody scaffold). To this end, we will develop new methodologies to significantly advance the fundamental knowledge about isolated picobodies.