De novo design protein binders for MBP and GST tags

Maltose-binding protein (MBP) and glutathione S-transferase (GST) are widely used solubility-enhancing protein tags, typically employed to address various issues related to protein expression and purification. The detection of these tags are usually achieved through binding of corresponding antibodies. Designing low-cost binders as alternatives to antibodies is of great significance. This study employed a de novo design approach, starting with a large number of protein scaffolds and screening out 6 candidate binders targeting MBP and 4 candidate binders targeting GST based on scoring functions. Flow cytometry low-affinity selection and biolayer interferometry (BLI) quantitative results showed that MBP and GST can interact strongly with one or several binders, exhibiting nanomolar binding. Among them, LZMB3 has a binding dissociation constant (K_D) of 54.05 ± 1.46 nM, while LJGB3 and LJGB4 have K_D values of 105.4 ± 1.812 nM and 437.9 ± 17.69 nM, respectively.