CRD_SAT under Heat: Balancing Stability, Affinity, and Functional Utility of Computationally Designed Tag Variants

In general, the easier and cheaper the expression and purification processes are, the more profitable the production of a recombinant protein of interest is, especially in the industrial world. Previously, we have developed the lectinic CRD_SAT tag that we demonstrated is efficient at cost-effectively purifying passenger proteins. It also has the advantage of being quite small and limiting steric hindrance upon release by protease cleavage. Here, we used protein sequence optimization to design highly thermostable versions of CRD_SAT and showed that the midpoint denaturation temperature could be increased from 55.8 to 92.2 °C. In fact, our variants (called CRD_VLs) possess the ability to support a heating step during the purification process, which represents an easy way to eliminate thermolabile proteins coming from the host cells when the recombinant proteins are produced in bacteria. To challenge our CRD_VLs, we fused them to a PET hydrolase exhibiting promising industrial activity at 70 °C and showed that CRD_VL-tagged enzymes remain active, even after heating, with a balancing effect due to the CRD_VL internal mutations. Surprisingly, whereas mutations in CRD_VLs were introduced far from the d-lactose binding site, we also showed that the affinity toward the disaccharide was clearly improved in the variants to reach a dissociation constant (K_d) of around 30 μM (the K_d of the CRD_SAT being measured at around 90 μM), paving the way for the use of our tag in applications such as lectin-based affinity enrichment or those requiring few, cheap, and simple purification steps.