Protein Ligation‐Assisted Reconstitution of Split HRP Fragments for Facile Production of HRP Fusion Proteins in E. coli

• Published in: Chembiochem : a European journal of chemical biology Vol. 24; no. 11; pp. e202200700 - n/a
• Main Authors: Heo, Seungnyeong, Baek, Jonghwi, Bae, Juhyun, Seo, Bo Am, Kim, Seongjun, Jeong, Subin, Kim, Suhyun, Ryu, Yiseul, Lee, Joong‐jae
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 01.06.2023
• Subjects: Bacteria; Biological activity; Biosensors; Chemical reactions; Chemical synthesis; Cytoplasm; E coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fine chemicals; Fragments; fusion protein; Horseradish peroxidase; Horseradish Peroxidase - metabolism; irreversible reconstitution; Peroxidase; protein ligation; Proteins; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; split protein; fusion protein; irreversible reconstitution; protein ligation; split protein; horseradish peroxidase
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.202200700

Horseradish peroxidase (HRP) is a pivotal biocatalyst for biosensor development and fine chemical synthesis. HRP proteins are mostly extracted and purified from the roots of horseradish because the solubility and productivity of recombinant HRP in bacteria are significantly low. In this study, we investigate the reconstitution system of split HRP fragments to improve its soluble expression levels in E. coli allowing the cost‐effective production of bioactive HRPs. To promote the effective association between two HRP fragments (HRPn and HRPc), we exploit SpyTag‐SpyCatcher chemistry, a versatile protein coupling method with high affinity and selectivity. Each HRP fragment was genetically fused with SpyTag and SpyCatcher, respectively, exhibiting soluble expression in the E. coli cytoplasm. The engineered split HRPs were effectively and irreversibly reconstituted into a biologically active and stable assembly that can catalyze intrinsic enzymatic reactions. Compared to the chaperone co‐expression system, our approach shows that the production yield of soluble HRP is comparable, but the purity of the final product is relatively high. Therefore, our results can be applied to the high‐yield production of recombinant HRP variants and other difficult‐to‐express proteins in bacteria without complex downstream processes. The combination of a split protein system and protein ligation using SpyTag‐SpyCatcher chemistry enables the efficient production of covalently reconstituted, biologically functional HRP‐fusion proteins. This strategy can facilitate the construction of various kinds of stable bio‐composites based on split protein fragments in an irreversible manner.