Antibodies Purification Using ELP-zz Domain Fusions
Antibodies are immune system&ndashrelated proteins called immunoglobulins (IgGs) which have applications for medical diagnostics and research. However, their purification from different sources has always been a challenge because of low antibody concentration and higher purity requirements for usage. The unique capability of Elastin like Proteins (ELPs) to reversibly precipitate at a relatively modest temperature has been utilized for purification of antibodies. This feature of ELPs to purify antibodies has already been explored using larger fusion domains such as Protein L and Protein G. However the usage of larger Protein G/L fusions with ELP resulted in 10&ndashfold lower expression when compare to ELP or ELP fusions with shorter peptides.
In the current work, ELP fusions with a small IgG&ndashbinding peptide i.e. zz domain (a synthetic IgG binding domain derived from the Staphylococcus aureus protein A) were generated. The production of ELP[VPGVG]&ndash78&ndashzz fusion in E. coli ( &sim 500 mg/L) was found to be five-fold higher than ELP[VPGVG]&ndash78&ndashProL (&sim100 mg/L). In addition, the ELP[VPGVG]&ndash78&ndashzz fusion showed excellent binding affinity toward human, mouse, and rabbit IgGs, enabling simple purification of the different antibodies by reversible thermal precipitation. In order to recover antibody from the ELP&ndashzz&ndashIgG complex, different elution conditions were investigated. Close to 90% recovery was achieved using 0.5 M arginine pH 3.8 buffer.
To further increase the production of ELP-zz fusion protein, three different ELP domains (VPGXG)&ndash40 (where X= K:V:F=1:8:1); (VPGXG)&ndash60 (where X= K:V:F=1:8:1), (VPGXG)&ndash80 (where X= K:V:F=1:8:1), were generated. Production of ELP(KV8F)&ndashzz fusions were increased by two fold, while maintaining similar binding affinity for IgGs. Due to its high level production and affinity for different IgGs, we believe that these ELP-zz fusions will be useful as an economical, highly efficient, and universal platform for the purification of antibodies.