Expression and purification of recombinant Protein L variants in Escherichia coli.

Protein L is a κ light-chain-binding protein, which is widely used as an affinity ligand for antibody purification and characterization. However, manufacturable recombinant Protein L ligands remain insufficiently studied, particularly regarding how domain architecture affects production, processability, and product quality. In the current study, we designed, expressed and purified three recombinant Protein L variants with distinct domain architectures. This allowed the influence of domain architecture on manufacturability, stability, and downstream functionality to be explored. While differences in intermediate purity and recovery were observed among the three different variants, the optimized process allowed highly purified recombinant Protein L to be generated. As a functional validation, one of the purified variants (i.e., rPL02) was immobilized onto a membrane support, which was used to purify a κ light chain-containing single-chain variable fragment (scFv). The Protein L membrane device not only captured the target scFv but also removed the product-related aggregates under the tested conditions. In conclusion, the current work established a practical platform for producing functional recombinant Protein L. Meanwhile, it provided valuable information for future development of manufacturable recombinant Protein L for affinity media.