Antibody variable domains represent potential structural models for the rational design of therapeutic molecules that bind cellular proteins with high affinity and specificity. The Activating Transcription Factor 1(ATF1)/Cyclic AMP Response Element Binding Protein (CREB) family of transcription factors are particularly relevant targets due to their strong association with melanoma and clear cell sarcoma. A single-chain antibody fragment (scFv), scFv41.4, has been shown to disrupt the binding of ATF1/CREB to cyclic-AMP response elements (CRE) in vitro. Functional studies have verified that deletion of the light chain did not result in reduced inhibitory activity. Purified scFv41.4 expressed in Escherichia coli BL21 cells, was prepared to aid in further investigations of structure and binding interactions.