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One-step purification and immobilization of recombinant proteins using SpyTag/SpyCatcher chemistry

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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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References

  • Abian O, Grazu V, Hermoso J, Gonzalez R, Garcia JL, Fernandez-Lafuente R, Guisan JM (2004) Stabilization of penicillin G acylase from Escherichia coli: site-directed mutagenesis of the protein surface to increase multipoint covalent attachment. Appl Environ Microb 70:1249–1251

    Article  CAS  Google Scholar 

  • Adriano WS, Silva JA, Giordano RLC, Gonçalves LRB (2005) Stabilization of penicillin G acylase by immobilization on glutaraldehyde-activated chitosan. Braz J Chem Eng 22:529–538

    Article  CAS  Google Scholar 

  • Anuar INK, Banerjee A, Keeble AH, Carella A, Nikov GI, Howarth M (2019) Spy&Go purification of SpyTag-proteins using pseudo-SpyCatcher to access an oligomerization toolbox. Nat Commun 10:1–13

    Article  Google Scholar 

  • Ansari SA, Husain Q (2012) Potential applications of enzymes immobilized on/in nano materials: a review. Biotechnol Adv 30:512–523

    Article  CAS  Google Scholar 

  • Ardao I, Benaiges MD, Caminal G, Alvaro G, (2006) One step purification–immobilization of fuculose-1-phosphate aldolase, a class II DHAP dependent aldolase, by using metal-chelate supports. Enzyme Microb Technol 39:22–27

    Article  CAS  Google Scholar 

  • Barbosa O, Ortiz C, Berenguer-Murcia A, Torres R, Rodrigues RC, Fernandez-Lafuente R (2015) Strategies for the one-step immobilization-purification of enzymes as industrial biocatalysts. Biotechnol Adv 33:435–456

    Article  CAS  Google Scholar 

  • Chern JT, Chao YP (2005) Chitin-binding domain based immobilization of d-hydantoinase. Biotechnology 117:267–275

    CAS  Google Scholar 

  • Engler C, Marillonnet S (2014) Golden gate cloning, vol 1116. Humana Press, Totowa, pp 119–131

    Google Scholar 

  • Gaberc-Porekar V, Menart V (2001) Perspectives of immobilized-metal affinity chromatography. J Biochem Bioph Meth 49:335–360

    Article  CAS  Google Scholar 

  • Gao X, Fang J, Xue B, Fu L, Li H (2016) Engineering protein hydrogels using SpyCatcher-SpyTag chemistry. Biomacromol 17:2812–2819

    Article  CAS  Google Scholar 

  • Li L, Fierer JO, Rapoport TA, Howarth M (2014) Structural analysis and optimization of the covalent association between SpyCatcher and a peptide tag. J Mol Biol 426:309–317

    Article  CAS  Google Scholar 

  • Linder M, Nevanen T, Söderholm L, Bengs O, Teeri TT (1999) Improved immobilization of fusion proteins via cellulose-binding domains. Biotechnol Bioeng 60:642–647

    Article  Google Scholar 

  • Liu X, Yang X, Yang Z, Luo J, Tian X, Liu K, Sun F (2018) Versatile engineered protein hydrogels enabling decoupled mechanical and biochemical tuning for cell adhesion and neurite growth. ACS Appl Nano Mater 1:1579–1585

    Article  CAS  Google Scholar 

  • Luo H, Zhao H, Chang Y, Wang Q, Yu H, Shen Z (2015) Oriented immobilization and characterization of a poly-lysine-tagged cephalosporin C acylase on glyoxyl agarose support. Appl Biochem Biotechnol 175:2114–2123

    Article  CAS  Google Scholar 

  • Pedroche J, del Mar YM, Mateo C, Fernández-Lafuente R, Girón-Calle J, Alaiz M, Vioque J, Guisán JM, Millán F (2007) Effect of the support and experimental conditions in the intensity of the multipoint covalent attachment of proteins on glyoxyl-agarose supports: correlation between enzyme-support linkages and thermal stability. Enzyme Microb Technol 40:1160–1166

    Article  CAS  Google Scholar 

  • Reddington SC, Howarth MR (2015) Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher. Curr Opin Chem Biol 29:94–99

    Article  CAS  Google Scholar 

  • Rehm FBH, Chen S, Rehm BHA (2018) Bioengineering toward direct production of immobilized enzymes: a paradigm shift in biocatalyst design. Bioengineered 9:6–11

    Article  Google Scholar 

  • Sanchis J, Fernandez L, Carballeira JD, Drone J, Gumulya Y, Hobenreich H, Kahakeaw D, Kille S, Lohmer R, Peyralans JJ, Podtetenieff J, Prasad S, Soni P, Taglieber A, Wu S, Zilly FE, Reetz MT (2008) Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates. Appl Microbiol Biotechnol 81:387–397

    Article  CAS  Google Scholar 

  • Santos JCSD, Barbosa O, Ortiz C, Berenguer-Murcia A, Rodrigues RC, Fernandez-Lafuente R (2015) Importance of the support properties for immobilization or purification of enzymes. ChemCatChem 7:2413–2432

    Article  Google Scholar 

  • Scaramozzino F, Estruch I, Rossolillo P, Terreni M, Albertini AM (2005) Improvement of catalytic properties of Escherichia coli penicillin G acylase immobilized on glyoxyl agarose by addition of a six-amino-acid tag. Appl Environ Microbiol 71:8937–8940

    Article  CAS  Google Scholar 

  • Si M, Xu Q, Jiang L, Huang H (2016) SpyTag/SpyCatcher cyclization enhances the thermostability of firefly luciferase. PLoS ONE 11:e0162318

    Article  Google Scholar 

  • Thrane S, Janitzek CM, Matondo S, Resende M, Gustavsson T, De Jongh WA, Sauerwein R, Schiller JT, Nielsen MA, Theander TG, Salanti A, Sander AF (2016) Bacterial superglue enables easy development of efficient virus-like particle based vaccines. J Nanobiotechnol 14:1–16

    Article  Google Scholar 

  • Wang Y, Tian J, Xiao Y, Wang Y, Luo H (2019) SpyTag/SpyCatcher cyclization enhances the thermostability and organic solvent tolerance of l-phenylalanine aldolase. Biotechnol Lett 41:987–994

    Article  CAS  Google Scholar 

  • Yin L, Guo X, Liu L, Zhang Y, Feng Y (2018) Self-assembled multimeric-enzyme nanoreactor for robust and efficient biocatalysis. ACS Biomater Sci Eng 4:2095–2099

    Article  CAS  Google Scholar 

  • Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci USA 109:E690–E697

    Article  CAS  Google Scholar 

  • Zhou YB, Cao JW, Sun XB, Wang H, Gao DY, Li YN, Wu KY, Wang JK, Qian GY, Wang Q (2020) Enhanced stability of a rumen-derived xylanase using SpyTag/SpyCatcher cyclization. World J Microb Biot 36:1–11

    Article  CAS  Google Scholar 

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Supplementary information

Supplementary Table S1—Primers used in the plasmid construction.

Funding

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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Correspondence to Yanhong Chang or Hui Luo.

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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

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