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One-step non-chromatography purification of a low abundant fucosylated protein from complex plant crude extract

School of Chemical and Biomolecular Engineering Georgia Institute of Technology, Atlanta, GA 30332-0100, USA

Special Issues: Microbial biotechnology

Effective methods for isolation and purification of glycoproteins and other glycoconjugates are important to biopharmaceutical industry and diagnostic industry. They are also critical to an emerging field of glycoproteomics. In this work, we applied the newly-developed affinity ligand, a fusion protein of elastic like polymer (ELP) and a bacterial lectin, in an affinity precipitation process to purify soybean peroxidase (SBP) based on the presence of fucoseon the protein surface. We addressed, in particular, the challenge of purifying a low abundant protein from a complex dilute crude plant extract. The novel affinity precipitation developed in this work was very promising. One step binding and precipitation resulted in >95% recovery yield directly from crude extract and a 22.7 fold purification, giving a specific activity of 420 U/mg. The SBP isolated using this affinity precipitation meets or exceeds the quality specifications of reagent grade products by Sigma. We showed that the recovery yield had a strong dependence on the molar ratio of ligand to target fucosylated protein, with a ratio of three giving nearly full recovery, which could be predicted based on the total fucose content per protein molecule and the number of binding site per ligand molecule. We additionally developed a method of ligand regeneration and investigated its reuse. A simple wash with pH buffer was shown to be effective to regenerate the binding capacity for the ligand, and the ligand could be used for 10 times, giving an averaged 80% isolation yield based on initial input of soybean peroxidase. Taken together, an effective method of affinity precipitation was developed, which could be used to enrich a low abundant target glycoprotein from a complex mixture with a high recovery yield. The high selectivity for fucosylated protein and its ease of operation make this method particularly useful for purification of low abundant glycoprotein from natural sources. This work establishes a non-chromatography glycoform-specific purification method and extends the useful ELP-based affinity precipitation to glycoproteins.
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Keywords affinity precipitation; bacterial lectins; fucosylated proteins; glycoprotein purification; soybean peroxidase

Citation: Lindsay Arnold, Rachel Chen. One-step non-chromatography purification of a low abundant fucosylated protein from complex plant crude extract. AIMS Bioengineering, 2015, 2(3): 249-263. doi: 10.3934/bioeng.2015.3.249

References

  • 1. Ghazarian H, Idoni B, Oppenheimer SB. (2011) A glycobiology review: carbohydrates, lectins and implications in cancer therapeutics. Acta Histochem 113:236-247.    
  • 2. Wiederschain GY (2013) Glycobiology: progress, problems, and perspectives. Biochem Biokhimiia 78: 679-696. 3. Varki A, Cummings RD, Esko JD, et al. (2009) Essentials of Glycobiology. 2nd ed. Cold Spring Harbor, editor. NY: Cold Spring Harbor Laboratory Press.    
  • 4. Ekins S, Xu JJ (2009) Drug Efficacy, Safety, and Biologics Discovery: Emerging Technologies and Tools. Hoboken, New Jersey: John Wiley & Sons, Inc.
  • 5. Jefferis R (2009) Glycosylation as a strategy to improve antibody-based therapeutics. Nat Rev Drug Discov 8: 226-234.    
  • 6. Nakano M, Kondo A, Kakehi K, et al. (2006) Glycomics - a new target for pharmaceuticals. Drug Discov Today Technol 3: 39-47.    
  • 7. Raman R, Venkataraman M, Ramakrishnan S, et al. (2006) Advancing glycomics: implementation strategies at the consortium for functional glycomics. Glycobiology 16: 82R-90R.    
  • 8. Taniguchi N, Ekuni A, Ko JH, et al. (2001) A glycomic approach to the identification and characterization of glycoprotein function in cells transfected with glycosyltransferase genes. Proteomics 1: 239-247.
  • 9. Gabius H-J, André S, Jiménez-Barbero J, et al. (2011) From lectin structure to functional glycomics: principles of the sugar code. Trends Biochem Sci 36: 298-313.    
  • 10. VanHaandel MJ, Sarabèr FC, Boersma MG, et al. (2000) Characterization of different commercial soybean peroxidase preparations and use of the enzyme for N-demethylation of methyl N-methylanthranilate To produce the food flavor methylanthranilate. J Agric Food Chem 48: 1949-1954.    
  • 11. Chattopadhyay K, Mazumdar S (2000) Structural and Conformational Stability of Horseradish Peroxidase: Effect of Temperature and pH. Biochemistry 39: 263-270.    
  • 12. Sakharov IY, Berlina AN, Zherdev AV, et al. (2010) Advantages of soybean peroxidase over horseradish peroxidase as the enzyme label in chemiluminescent enzyme-linked immunosorbent assay of sulfamethoxypyridazine. J Agric Food Chem 58: 3284-3289.    
  • 13. Husain Q (2009) Peroxidase mediated decolorization and remediation of wastewater containing industrial dyes: a review. Rev Environ Sci Bio/Technology 9: 117-140.
  • 14. Mceldoon JP, Dordick JS (1996) Unusual Thermal Stability of Soybean Peroxidase. Biotechnol Prog 7938(pH 8): 555-558.
  • 15. Henriksen A, Mirza O, Indiani C, et al. (2001) Structure of soybean seed coat peroxidase: A plant peroxidase with unusual stability and haem-apoprotein interactions. Protein Sci 10: 108-115.
  • 16. Kamal JKA, Behere DV (2002) Thermal and Conformational Stability of Seed Coat Soybean Peroxidase. Biochemistry 41: 9034-9042.    
  • 17. Gillikin JW, Graham JS (1991) Purification and Developmental Analysis of the Major Anionic Peroxidase from the Seed Coat of Glycine max. Plant Physiol 96:214-220.
  • 18. Hailu G, Weersink A, Cahlík F (2010) Examining the Prospects for Commercialization of Soybean Peroxidase. AgBioForum 13: 263-273.
  • 19. Franco FL, Batista-Viera F, Carlsson J (2004) Preparation of high-density Concanavalin A adsorbent and its use for rapid, high-yield purification of peroxidase from horseradish roots. J Chromatogr B Analyt Technol Biomed Life Sci 803: 237-241.
  • 20. Jungbauer A (1993) Review Preparative chromatography of biomolecules. J Chromatogr 639: 3-16.    
  • 21. Patchornik G, Albeck A (2005) Free nonimmobilized ligands as a tool for purification of proteins. Bioconjug Chem 16: 1310-1315.    
  • 22. Lam SK, Ng TB (2011) Lectins: production and practical applications. Appl Microbiol Biotechnol 89: 45-55.    
  • 23. Lee LY, Hincapie M, Packer N, et al. (2012) An optimized approach for enrichment of glycoproteins from cell culture lysates using native multi-lectin affinity chromatography. J Sep Sci 35: 2445-2452.    
  • 24. Hilbrig F, Stocker G, Schläppi J-M, et al. (2006) Utilization of Group Specific Ligands in the Downstream Processing of Proteins by Affinity Precipitation. Food Bioprod Process 84: 28-36.
  • 25. Gottschalk U (2005) Downstream Processing of Monoclonal Antibodies: from High Dilution to High Purity. BioPharm Int 18: 42-58.
  • 26. Linné-Larsson E, Galaev I, Lindahl L, et al. (1996) Affinity precipitation of Concanavalin A with p-aminophenyl- α-D-glucopyranoside modified Eudragit S-100. Bioseparation 6: 273-282.
  • 27. Urry DW, Gowda DC, Parker TM, et al. (1992) Hydrophobicity scale for proteins based on inverse temperature transitions. Biopolymers 32: 1243-1250.    
  • 28. Fong B, Wu W, Wood DW (2009) Optimization of ELP-intein mediated protein purification by salt substitution. Protein Expr Purif 66: 198-202.
  • 29. Madan B, Chaudhary G, Cramer SM, et al. (2013) ELP-z and ELP-zz capturing scaffolds for the purification of immunoglobulins by affinity precipitation. J Biotechnol 163: 10-16.    
  • 30. Arnold L, Chen R (2014) Novel thermo-responsive fucose binding ligands for glycoprotein purification by affinity precipitation. Biotechnol Bioeng 111: 413-417.    
  • 31. Liu J, Zhang Y, Qiu L, et al. (2005) A Novel Process of Purifying Soybean Hull Peroxidase. Chem Biochem Eng Q 19: 199-205.
  • 32. Gray JS, Yang BY, Hull SR, et al. (1996) The glycans of soybean peroxidase. Glycobiology. 6: 23-32.    
  • 33. Sessa DJ (2004) Processing of soybean hulls to enhance the distribution and extraction of value-added proteins. J Sci Food Agric 84: 75-82.    
  • 34. Meyer DE, Chilkoti A (1999) Purification of recombinant proteins by fusion with thermally-responsive polypeptides. Nat Biotechnol 17: 1112-1115.    
  • 35. Meyer DE, Trabbic-Carlson K, Chilkoti A (2001) Protein purification by fusion with an environmentally responsive elastin-like polypeptide: effect of polypeptide length on the purification of thioredoxin. Biotechnol Prog 17: 720-728.    
  • 36. Banki MR, Feng LA, Wood DW (2005) Simple bioseparations using self-cleaving elastin-like polypeptide tags. Nat Methods 2: 659-661.    
  • 37. Wood DW (2010) Non-Chromatographic Recombinant Protein Purification by Self-Cleaving Purification Tags. Sep Sci Technol 45: 2345-2357.
  • 38. Ge X, Yang DSC, Trabbic-carlson K, et al. (2005) Self-Cleavable Stimulus Responsive Tags for Protein Purification without Chromatography. J Am Chem Soc 127: 11228-11229.    
  • 39. Tian L, Sun SSM (2011) A cost-effective ELP-intein coupling system for recombinant protein purification from plant production platform. PLoS One 6: e24183.    
  • 40. Shimazu M, Mulchandani A, Chen W (2003) Thermally triggered purification and immobilization of elastin-OPH fusions. Biotechnol Bioeng 81: 74-79.    
  • 41. Liu F, Tsai S-L, Madan B, et al. (2012) Engineering a high-affinity scaffold for non-chromatographic protein purification via intein-mediated cleavage. Biotechnol Bioeng 109: 2829-2835.    
  • 42. Kostlanova N, Mitchell EP, Lortat-Jacob H, et al. (2005) The fucose-binding lectin from Ralstonia solanacearum - A new type of beta-propeller architecture formed by oligomerization and interacting with fucoside, fucosyllactose, and plant xyloglucan. J Biol Chem 280: 27839-27849.
  • 43. Mammen M, Choi S-K, Whitesides GM (1998) Polyvalent Interactions in Biological Systems: Implications for Design and Use of Multivalent Ligands and Inhibitors. Angew Chemie Int Ed 37: 2754-2794.
  • 44. Wang L-X, Amin MN (2014) Chemical and chemoenzymatic synthesis of glycoproteins for deciphering functions. Chem Biol 21: 51-66.    
  • 45. Sheth RD, Jin M, Bhut BV, et al. (2014) Affinity precipitation of a monoclonal antibody from an industrial harvest feedstock using an ELP-Z stimuli responsive biopolymer. Biotechnol Bioeng 111: 1595-1603.    

 

This article has been cited by

  • 1. Lindsay Arnold, Tian-Bo Yang, Rachel Chen, A thermal responsive affinity ligand for precipitation of sialylated proteins, AIMS Bioengineering, 2016, 3, 1, 92, 10.3934/bioeng.2016.1.92
  • 2. Hanaa E.A. Amer, , Proteomics Technologies and Applications [Working Title], 2019, 10.5772/intechopen.86587

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Copyright Info: 2015, Rachel Chen, et al., licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution Licese (http://creativecommons.org/licenses/by/4.0)

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