SpyTag/Catcher chemistry induces the formation of active inclusion bodies in E. coli

• Published in: International journal of biological macromolecules 2022-02, Vol.199, p.358-371
• Main Authors: Dong, Wenge, Sun, Hongxu, Chen, Qiwei, Hou, Liangyu, Chang, Yanhong, Luo, Hui
• Format: Article
• Language: English
• Subjects: Active inclusion bodies; Biocatalyst; Cross-linked enzyme aggregates; Cross-Linking Reagents - chemistry; Enzyme Stability; Enzymes, Immobilized - chemistry; Escherichia coli - genetics; Escherichia coli - metabolism; Glutaral - metabolism; Inclusion Bodies; Multimeric enzyme; Protein Aggregates; Proteins - metabolism; SpyTag/Catcher
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2022.01.018

SpyTag/Catcher chemistry is usually applied to engineer robust enzymes via head-to-tail cyclization using spontaneous intramolecular isopeptide bond formation. However, the SpyTag/Catcher induced intercellular protein assembly in vivo cannot be ignored. It was found that some active inclusion bodies had generated to different proportions in the expression of six SpyTag/Catcher labeled proteins (CatIBs-STCProtein). Some factors that may affect the formation of CatIBs-STCProtein were discussed, and the subunit quantities were found to be strongly positively related to the formation of protein aggregates. Approximately 85.44% of the activity of the octameric protein leucine dehydrogenase (LDH) was expressed in aggregates, while the activity of the monomeric protein green fluorescence protein (GFP) in aggregates was 12.51%. The results indicated that SpyTag/Catcher can be used to form protein aggregates in E. coli. To facilitate the advantages of CatIBs-STCProtein, we took the CatIBs-STCLDH as an example and further chemically cross-linked with glutaraldehyde to obtain novel cross-linked enzyme aggregates (CLEAs-CatIBs-STCLDH). CLEAs-CatIBs-STCLDH had good thermal stability and organic solvents stability, and its activity remained 51.03% after incubation at 60 °C for 100 mins. Moreover, the crosslinked CatIBs-STCLDH also showed superior stability over traditional CLEAs, and its activity remained 98.70% after 10 cycles of catalysis. [Display omitted] •Protein can be cross-linked intracellularly by fusing the label of SpyTag/Catcher.•Cross-linked proteins form aggregates named catalytically active inclusion bodies.•Aggregation rate of protein increases with the number of protein subunits.•Stability of aggregates was enhanced after cross-linking with glutaraldehyde.