# Engineering of an Fc-specific monovalent protein G for the light-controlled affinity purification of antibodies

## TL;DR

Scientists engineered a modified protein G that can purify antibodies using light, avoiding harsh conditions and unwanted cross-linking.

## Contribution

A novel monovalent protein G mutant (ProtGN478R) was developed for light-controlled antibody purification without cross-linking.

## Key findings

- ProtGN478R eliminates antibody precipitation by losing Fab-binding activity while retaining Fc-binding affinity.
- Azo-ProtGN478R enables single-step antibody purification from complex mixtures using UV-A light.
- The modified protein avoids harsh pH shifts during elution via light-triggered isomerization.

## Abstract

Like other widely applied bacterial surface receptor proteins for immunoglobulins (Igs), such as protein A and protein L, the Ig-binding domain of protein G (ProtG) has dual binding activity. ProtG can independently associate both with the Fc region of an antibody (mAb) and with its Fab and, thus, provoke cross-linking if applied in solution. Indeed, we observed pronounced precipitation activity when using ProtG equipped with the Azo-tag as a small adapter molecule for the light-controlled affinity purification of mAbs. We demonstrate that this undesired precipitation phenomenon follows the classical Heidelberger-Kendall curve. Furthermore, we describe a mutant of ProtG in which Asn478 at the interface with the Fab is replaced by Arg, which results in the effective loss of this secondary binding activity while maintaining high affinity towards the Ig Fc region. ProtGN478R no longer induces precipitation when mixed with a series of medically relevant mAbs. Hence, Azo-ProtGN478R can be applied as a convenient molecular tool to isolate antibodies from cell culture medium—even with a high content of albumin—in a single step via Excitography. In this technique, elution is triggered by trans → cis isomerisation of the Azo-tag upon illumination with mild UV-A light and a harsh pH shift is avoided.

The online version contains supplementary material available at 10.1038/s41598-025-25894-5.