Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Metal-chelate affinity precipitation of proteins using responsive polymers

Abstract

Affinity precipitation of proteins uses polymers capable of reversible soluble-insoluble transitions in response to small environmental changes (temperature, pH or solvent composition). Here we describe protocols for (i) the synthesis of responsive polymers with specific affinity to target proteins and (ii) the purification of proteins using these polymers. The purification is based on precipitation of the affinity complex between the protein and the polymer, which is induced by environmental changes. This separation strategy is simpler and more cost effective than conventional affinity column chromatography. Specifically, we describe the synthesis of thermoresponsive 1-vinylimidazole:N-isopropylacrylamide copolymers. The whole procedure takes 2–3 h when applied to purification of recombinant His-tag proteins or proteins with natural metal binding groups by means of metal chelate affinity precipitation. Optimization of the polymer composition and the type of chelating ions allows for target protein yields of 80% and higher.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Schematic of metal chelate affinity precipitation of proteins.
Figure 2: Structure of a 1-vinylimidazole:N-isopropylacrylamine copolymer.

References

  1. Wilchek, M. & Jakoby W.B. The literature on affinity chromatography. Meth. Enzymol. 34, 3–10 (1974).

    CAS  Article  Google Scholar 

  2. Heskins, M., Guillet, J.E. Solution properties of poly(N-isopropylacrylamide). J. Macromol. Sci. Chem. A2, 1441–1455 (1968).

    Article  Google Scholar 

  3. Galaev, I.Y. & Mattiasson, B. “Smart” polymers and what they can do in biotechnology and medicine. Trends Biotechnol. 17, 335–340 (1999).

    CAS  Article  Google Scholar 

  4. Guoqiang, D., Benhura, M.A.N., Kaul, R. & Mattiasson B. Affinity thermoprecipitation of yeast alcohol dehydrogenase through metal ion-promoted binding with Eudragit-bound Cibacron Blue 3GA. Biotechnol. Prog. 11, 187–193 (1995).

    CAS  Article  Google Scholar 

  5. Smith, M.C., Furman, T.C., Ingolia, T. D. & Pidgeon, C. Chelating peptide immobilized metal ion affinity chromatography. J. Biol. Chem. 263, 7211–7215 (1988).

    CAS  PubMed  Google Scholar 

  6. Kumar, A., Wahlund, P.-O., Kepka, C., Galaev, I.Y. & Mattiasson, B. Purification of histidine-tagged single chain Fv-antibody fragments by metal chelate affinity precipitation using thermo-responsive copolymers. Biotechnol. Bioeng. 84, 495–503 (2003).

    Article  Google Scholar 

  7. Hochuli, E., Bannwarth, W., Döbeli, H., Gentz, R. & Stüber, D. Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Bio/Technol 6, 1321–1325 (1988).

    CAS  Google Scholar 

  8. Hilbrig, F., Freitag, R. Protein purification by affinity precipitation. J. Chromatogr. B. 790, 79–90 (2003).

    CAS  Article  Google Scholar 

  9. Sinquin, A., Hubert P., Dellacherie E. Amphiphilic derivatives of alginate–evidence for intramolecular and intermolecular hydrophobic associations in aqueous solution. Langmuir 9, 3334–3337 (1993).

    CAS  Article  Google Scholar 

  10. Branham, K.D., Snowden, H.S., McCormick, C.L. Water-soluble copolymers. 64. Effects of pH and composition on associative properties of amphiphilic acrylamide/acrylic acid terpolymers. Macromolecules 29, 254–262 (1996).

    CAS  Article  Google Scholar 

  11. Linné-Larsson, E., Galaev, I.Y., Lindahl, L. & Mattiasson, B. Affinity precipitation of concanavalin A with p-amino-α-D-glycopyranoside modified Eudragit S-100. Initial complex formation and build-up of the precipitate. Bioseparation 6, 273–282 (1996).

    Google Scholar 

  12. Lehmann, K., Petereit, H.U. & Dreher, D. Fast disintegrating controlled-release tablets from coated particles. Pharmazeutische Industrie 55, 940–947 (1993).

    CAS  Google Scholar 

  13. Taniguchi, M., Kobayashi, M., Natssui, K. & Fujii, M. Purfification of staphylococcal protein A by affinity precipitation using a reversibly soluble-insoluble polymer with human IgG as a ligand. J. Ferment. Bioeng. 68, 32–36 (1989).

    CAS  Article  Google Scholar 

  14. Senstad, C. & Mattiasson, B. Affinity precipitation using chitosan as a ligand carrier. Biotechnol. Bioeng. 33, 216–220 (1989).

    CAS  Article  Google Scholar 

  15. Senstad, C. & Mattiasson, B. . Purification of wheat germ agglutinin using affinity flocculation with chitosan and a subsequent centrifugation or flotation step. Biotechnol. Bioeng. 34, 387–393 (1989).

    CAS  Article  Google Scholar 

  16. Tyagi, R., Kumar, A., Sardar, M., Kumar, S. & Gupta, M.N. Chitosan as an affinity macroligand for precipitation of N-acetyl glucosamine binding proteins/enzymes. Isol. Purif. 2, 217–226 (1996).

    CAS  Google Scholar 

  17. Guoqiang, D., Kaul, R. & Mattiasson, B. Purification of Lactobacillus bulgaricus D-lactate dehydrogenase by precipitation with an anionic polymer. Bioseparation 3, 333–341 (1993).

    Google Scholar 

  18. Idziak, I., Avoce, D., Lessard, D., Gravel, D. & Zhu, X.X. . Thermosensitivity of aqueous solutions of poly(N,N-diethylacrylamide). Macromolecules 32, 1260–1263 (1999).

    CAS  Article  Google Scholar 

  19. Lozinsky, V.I. et al. Synthesis of N-vinylcaprolactam polymers in water-containing media. Polymer 41, 6507–6518 (2000).

    CAS  Article  Google Scholar 

  20. Schild, H.G. Poly(N-isopropylacrylamide): experiment, theory and applications. Prog. Polym. Sci. 17, 163–249 (1992).

    CAS  Article  Google Scholar 

  21. Maeda, M., Nishimura, C., Inenaga, A. & Takagi, M. Modification of DNA with poly(N-isopropylacrylamide) for thermally induced affinity precipitation. Reactive Funct. Polym. 21, 27–35 (1993).

    CAS  Article  Google Scholar 

  22. Liu, F. et al. Development of a polymer-enzyme immunoassay method and its application. Biotechnol. Appl. Biochem. 21, 257–264 (1995).

    CAS  PubMed  Google Scholar 

  23. Umeno, D., Mori, T. & Maeda, M. Single stranded DNA-poly(N-isopropyl-acrylamide) conjugate for affinity precipitation separation of oligonucleotides. Chem. Commun. 14, 1433–1434 (1998).

    Article  Google Scholar 

  24. Stiborova, H., Kostal, J., Mulchandani, A. & Chen, W. One-step metal-affinity purification of histidine-tagged proteins by temperature-triggered precipitation. Biotechnol. Bioeng. 82, 605–611 (2003)

    CAS  Article  Google Scholar 

  25. Linné., E., Garg, N., Kaul, R. & Mattiasson, B. Evaluation of alginate as a carrier in affinity precipitation. Biotechnol. Appl. Biochem. 16, 48–56 (1992).

    Google Scholar 

  26. Roy, I. & Gupta, M.N. kappa-Carrageenan as a new smart macroaffinity ligand for the purification of pullulanase. J. Chromatogr . A 998, 103–108 (2003).

    CAS  Google Scholar 

  27. Bradshaw, A.P. & Sturgeon, R.J. The synthesis of soluble polymer-ligand complexes for affinity precipitation studies. Biotechnol. Tech. 4, 254–263 (1990).

    Article  Google Scholar 

  28. Ivanov, A.E., Shiomori, K., Kawano, Y., Galaev, I.Y. & Mattiasson, B. Effects of polyols, saccharides and glycoproteins on thermoprecipitation of phenylboronate-containing polymers. Biomacromolecules 7, 1017–1024 (2006).

    CAS  Article  Google Scholar 

  29. Galaev, I.Y. & Mattiasson, B. Affinity thermoprecipitation of trypsin using soybean trypsin inhibitor conjugated with a thermo-reactive polymer, poly(N-vinyl caprolactam). Biotechnol. Tech. 6, 353–358 (1992).

    CAS  Article  Google Scholar 

  30. Chen, J.-P. & Jang, F.-L. Purification of trypsin by affinity precipitation combining with aqueous two-phase extraction. Biotechnol. Tech. 9, 461–466 (1995).

    CAS  Article  Google Scholar 

  31. Kamihira, M., Kaul, R. & Mattiasson, B. Purification of recombinant protein A by aqueous two-phase extraction integrated with affinity precipitation. Biotechnol. Bioeng. 40, 1381–1387 (1992).

    CAS  Article  Google Scholar 

  32. Fong, R.B., Ding, Z.L., Long, C.J., Hoffman, A.S. & Stayton, P.S. Thermoprecipitation of streptavidin via oligonucleotide-mediated self-assembly with poly(N-isopropylacrylamide). Bioconjugate Chem. 10, 720–725 (1999).

    CAS  Article  Google Scholar 

  33. Linné-Larsson, E. & Mattiasson, B. Isolation of concanavalin A by affinity precipitation. Biotechnol. Techniq. 8, 51–56 (1994).

    Google Scholar 

  34. Nguyen, A.L. & Luong, J.H.T. Synthesis and application of water-soluble reactive polymers for purification and immobilization of biomolecules. Biotechnol. Bioeng. 34, 1186–1190 (1989).

    CAS  Article  Google Scholar 

  35. Kumar, A., Khalil, A.A.M., Galaev, I.Y. & Mattiasson, B. Metal chelate affinity precipitation: purification of (His)6-tagged lactate dehydrogenase using poly(vinylimidazole-co-N-isopropylacrylamide) copolymers. Enzyme Microb. Technol. 33, 113–117 (2003).

    CAS  Article  Google Scholar 

  36. Porath, J., Carlsson, J., Olsson, J. & Belfrage, G. Metal chelate affinity chromatography: a new approach to protein fractionation. Nature 258, 598–599 (1975).

    CAS  Article  Google Scholar 

  37. Arnold, F. H. Metal-affinity separations: a new dimension in protein processing. Bio/technology 9, 151–156 (1991).

    CAS  PubMed  Google Scholar 

  38. Galaev, I.Y., Kumar, A., Agarwal, R., Gupta, M.N. & Mattiasson, B. Imidazole: a new ligand for metal affinity precipitation. Precipitation of Kunitz soybean trypsin inhibitor using Cu(II)-loaded copolymers of 1-vinylimidazole with N-vinylcaprolactam or N-isopropylacrylamide. Appl. Biochem. Biotechnol. 68, 121–133 (1997)

    CAS  Article  Google Scholar 

  39. Kumar, A., Galaev, I.Y. & Mattiasson, B. Metal chelate affinity precipitation: a new approach to protein purification. Bioseparation 7, 185–194 (1999).

    Article  Google Scholar 

  40. Carter, S., Rimmer, S., Sturdy, A. & Webb, M. Highly branched stimuli responsive poly[(N-isopropylacrylamide)-co-(1,2-propandiol-3-methacrylate)]s with protein binding functionality. Macromol. Biosci. 5, 373–378 (2005).

    CAS  Article  Google Scholar 

  41. Balan, S. et al. Metal chelate affinity precipitation of RNA and purification of plasmid DNA. Biotechnol. Letters 25, 1111–1116 (2003).

    CAS  Article  Google Scholar 

  42. Costioli, M.D., Fisch, I., Garret-Flaudy, F., Hilbrig, F. & Freitag, R. DNA purification by triple-helix affinity precipitation. Biotechnol. Bioeng. 81, 535–545 (2003).

    CAS  Article  Google Scholar 

  43. Smith, D.B. & Johnson, K.S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67, 31–40 (1988).

    CAS  Article  Google Scholar 

  44. Maina, C.V. et al. An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose binding protein. Gene 74, 365–373 (1988).

    CAS  Article  Google Scholar 

  45. Ong, E. et al. The cellulose-binding domains of cellulases: tools for biotechnology. Trends. Biotechnol. 7, 239–243 (1989).

    CAS  Article  Google Scholar 

  46. Meyer, D.E. & Chilkoti, A. Purification of recombinant proteins by fusion with thermally reponsive polypeptides. Nature Biotechnol. 17, 1112–1115 (1999).

    CAS  Article  Google Scholar 

  47. Smith, P.K. et al. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85 (1985).

    CAS  Article  Google Scholar 

  48. Chen, G. & Hoffman, A. Synthesis of carboxylated poly(NIPAAm) oligomers and their application to form thermo-reversible polymer-enzyme conjugates. J. Biomater. Sci. Polymer Edn. 5, 371–382 (1994).

    Article  Google Scholar 

  49. Wahlund, P.-O., Galaev, I.Y., Kazakov, S.A., Lozinsky, V.I. & Mattiasson B. “Protein-like” copolymers: effect of polymer architecture on the performance in bioseparation process. Macromol. Biosci. 2, 33–42 (2002).

    CAS  Article  Google Scholar 

  50. Kumar, A., Galaev, I.Y. & Mattiasson, B. Affinity precipitation of amylase inhibitor from wheat meal by metal chelate affinity binding using Cu(II)-loaded copolymers of 1-vinylimidazole with N-isopropylacrylamide. Biotechnol. Bioeng. 59, 695–704 (1998).

    CAS  Article  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Bo Mattiasson.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Mattiasson, B., Kumar, A., Ivanov, A. et al. Metal-chelate affinity precipitation of proteins using responsive polymers. Nat Protoc 2, 213–220 (2007). https://doi.org/10.1038/nprot.2006.440

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.440

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing