Production, purification, and functional analysis of an artificial, multispecific protein based on an avidin scaffold

Abstract

Engineered, multispecific proteins show promise for many biological applications such as biosensors, immunotherapy targeting, and imaging and have advantages over multispecific antibodies since they are smaller and more stable. The tetrameric proteins avidin and streptavidin, known collectively as (strept)avidins, are already widely used in biomedical applications due to their strong biotin-binding affinity and their high stability. During this project, two forms of a previously-engineered artificial multispecific, single-chain protein based on an avidin scaffold (called scAvd1 and scAvd2) with binding specificities for the small ligands biotin, progesterone, and maleimide were produced in E. coli, and a purification protocol was developed for the proteins. These proteins are especially interesting because of the inter-changeability of the binding sites which gives them the potential to bind to any number of other small ligands depending on the binding sites genetically added. Purified proteins were examined using a variety of methods including enzyme-linked immunosorbent assay (ELISA) and spectrofluorometric binding analysis with progesterone and biotin. Further analyses using Octet and EnVision systems were performed using scAvd2 as a scaffold for enzymatic cascade reactions. Preliminary data from this project shows that the proteins are able to be produced in a functional form with the ability to bind to progesterone and maleimide and with a high affinity to biotin conserved despite modifications. Ultimately, scAvd could prove to be a useful tool for many research and medical applications as well as a way to overcome some issues with multispecific antibodies already being used in these applications. Furthermore, the developed proteins could be used in industry as a basis of enzymatic cascade reactions, increasing the stability of enzymes and bringing them spatially together, as demonstrated in the Octet measurements performed during this project. These multifunctional proteins also show promise as improved and novel tools for applications like surface biofunctionalization and targeted drug delivery. The proteins developed in this project with their binding specificities already show great promise for use in these and many more biotechnological applications, and as they are further optimized and modified, their scope of use will be even further expanded.