Proteins are naturally-occurring biopolymers that are useful in a wide range of therapeutic and imaging applications. Though natural proteins are comprised of just 20 monomeric amino acids, they can be post-translationally decorated to introduce expanded chemical functionality. Though several techniques have been developed, the majority do not provide site-specific modification nor stoichiometric conjugation necessary to preserve protein bioactivity. Additionally, proteins functionalized through these methods have proven difficult to purify after modification, thereby limiting potential clinical and industrial applications. Sortase-Tag Expressed Protein Ligation (STEPL) is a protein functionalization/purification method that solves many of these problems. This method allows functional probes to be site-specifically conjugated onto a protein, while simultaneously separating modified and unmodified proteins. Though this technique has been successfully demonstrated in the DeForest lab, a remaining challenge is to increase yield during protein expression. In our group, we exploit several microbiology techniques to optimize this process. Bacterial strain identity, growth conditions, reaction temperature, and protein solubility have all been shown to influence protein expression. Using Green Fluorescent Protein (GFP), we found that the STEPL yield increased when conducted for 4 hours at 37°C versus overnight at 18°C. This was determined through mass spectrometry, which showed hydrolyzed proteins after prolonged reaction. For some difficult to express proteins, including growth factors, the STEPL protocol resulted in a negligible protein yield. Through experimenting with a dialysis refolding protocol, functional growth factor Bone Morphogenic Protein-2 was produced at dramatically increased yields. Furthermore, experimenting with E. coli growth and STEPL reaction conditions has resulted in increased yield for many different functionalized proteins. Although progress has been made in producing functionalized protein with the STEPL method, yield remains below that obtained through other methods. Further experimentation to improve the robustness and yield of the STEPL protocol will increase the availability of functionalized proteins in biotherapeutics.