Video Article Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

doi:10.3791/3370 (2012). Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1. To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2. The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3. The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2. Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4, were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6. The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecula