Abstract
Based on the specific and spontaneous formation of isopeptide bonds by SpyCatcher/SpyTag, we have developed a one-step method for purification and immobilization of recombinant proteins. The procedure is to immobilize SpyCatcher on glyoxyl agarose gels, and then the SpyCatcher immobilisate can be used to immobilize the SpyTag-fused protein in the crude extract selectively. A mutant of SpyCatcher (mSC), in which a peptide (LysGlyLysGlyLysGly) was added to the C-terminus of SpyCatcher and three lysine residues around the SpyTag/SpyCatcher binding domain were replaced with arginine, was designed to improve the attachment of SpyCatcher to the support. Compared with wild-type SpyCatcher, mSC can be immobilized on the glyoxyl-agarose support more efficiently, which enables the obtained mSC derivative a high binding capacity of the SpyTag-fused protein. The results showed that the target proteins in the crude enzyme extract were purified and immobilized in one step, and the thermal stability of the immobilized target proteins was also remarkably improved.
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Supplementary information
Supplementary Table S1—Primers used in the plasmid construction.
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This work was financially supported by the National Natural Science Foundation of China (22078019; 21476025).
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JT, YC and HL conceived and designed the study. JT, RJ, WD, HS and YW performed the experiments and analyzed the data. JT wrote the paper. JT, YC and HL reviewed and edited the manuscript. All authors read and approved the manuscript.
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Tian, J., Jia, R., Wenge, D. et al. One-step purification and immobilization of recombinant proteins using SpyTag/SpyCatcher chemistry. Biotechnol Lett 43, 1075–1087 (2021). https://doi.org/10.1007/s10529-021-03098-x
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DOI: https://doi.org/10.1007/s10529-021-03098-x