De novo design protein binders for MBP and GST tags

Jinlong Zhou; Yue Xiao; Qian Tang; Yunjun Yan; Dongqi Liu; Houjin Zhang

doi:10.1016/j.bbrc.2025.151322

De novo design protein binders for MBP and GST tags

Biochem Biophys Res Commun. 2025 Feb 8:748:151322. doi: 10.1016/j.bbrc.2025.151322. Epub 2025 Jan 11.

Authors

Jinlong Zhou¹, Yue Xiao¹, Qian Tang², Yunjun Yan¹, Dongqi Liu³, Houjin Zhang⁴

Affiliations

¹ Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, MOE Key Laboratory of Molecular Biophysics, Wuhan, 430074, China.
² School of Environmental Science and Engineering, Huazhong University of Science and Technology, No. 1037 Luoyu Road, Wuhan, 430074, China.
³ School of Environmental Science and Engineering, Huazhong University of Science and Technology, No. 1037 Luoyu Road, Wuhan, 430074, China. Electronic address: liudongqi@hust.edu.cn.
⁴ Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, MOE Key Laboratory of Molecular Biophysics, Wuhan, 430074, China. Electronic address: hjzhang@hust.edu.cn.

PMID: 39827550
DOI: 10.1016/j.bbrc.2025.151322

Abstract

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.

Keywords: De novo design; Protein binders; Protein tags.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Glutathione Transferase* / chemistry
Glutathione Transferase* / metabolism
Maltose-Binding Proteins* / chemistry
Maltose-Binding Proteins* / metabolism
Protein Binding
Protein Engineering* / methods

Substances

Glutathione Transferase
Maltose-Binding Proteins