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

Area of Science:

• Biotechnology
• Protein Engineering
• Biochemistry

Background:

• Cost-effective and efficient recombinant protein production is crucial for industrial applications.
• Previously developed CRD_SAT tag offers efficient, cost-effective purification with minimal steric hindrance.
• Need for enhanced protein purification tags with improved stability for industrial processes.

Purpose of the Study:

• To design and engineer highly thermostable versions of the CRD_SAT tag.
• To evaluate the performance of these thermostable variants (CRD_VLs) in protein purification.
• To assess the impact of mutations on enzyme activity and binding affinity.

Main Methods:

• Protein sequence optimization was employed to enhance the thermostability of the CRD_SAT tag.
• Variants (CRD_VLs) were created and their midpoint denaturation temperatures were determined.
• CRD_VLs were fused to PET hydrolase, and their activity and stability after heat treatment were assessed.

Main Results:

• Midpoint denaturation temperature of CRD_VLs increased from 55.8 °C to 92.2 °C.
• CRD_VL-tagged enzymes retained activity after a heat purification step, demonstrating enhanced stability.
• CRD_VLs exhibited improved affinity for d-lactose, with a dissociation constant (K_d) of ~30 μM compared to ~90 μM for CRD_SAT.

Conclusions:

• Engineered CRD_VLs offer superior thermostability, enabling efficient purification of recombinant proteins via heat treatment.
• The improved stability and enhanced affinity of CRD_VLs facilitate cost-effective protein purification.
• CRD_VL tags show promise for applications in lectin-based affinity enrichment and simplified purification strategies.