Heterologous Expression and Optimized One-Step Separation of Levansucrase via Elastin-like Polypeptides Tagging System

  • Kang, Hye-Jin (Department of Biological Engineering, Inha University) ;
  • Kim, Jin-Hee (Department of Chemistry, Purdue University) ;
  • Chang, Woo-Jin (ERC for Advanced Bioseparation Technology, Inha University) ;
  • Kim, Eung-Soo (Department of Biological Engineering, Inha University) ;
  • Koo, Yoon-Mo (Department of Biological Engineering, Inha University)
  • Published : 2007.11.30

Abstract

Elastin-like polypeptides (ELPs) undergo a reversible inverse phase transition upon a change in temperature. This thermally triggered phase transition allows for a simple and rapid means of purifying a fusion protein. Recovery of ELPs-tagged fusion protein was easily achieved by aggregation, triggered either by raising temperature or by adding salt. In this study, levansucrase has been used as a model enzyme in the development of a simple one-step purification method using ELPs. The levansucrase gene cloned from Pseudomonas aurantiaca S-4380 was tagged with various sizes of ELPs to functionally express and optimize the purification of levansucrase. One of two ELPs, ELP[V-20] or ELP[V-40], was fused at the C-terminus of the levansucrase gene. A levansucrase-ELP fusion protein was expressed in Escherichia coli $DH5{\alpha}$ at $37^{\circ}C$ for 18 h. The molecular masses of levansucrase-ELP[V-20] and levansucrase-ELP[V-40] were determined as 56 kDa and 65 kDa, respectively. The phase transition of levansucrase-ELP[V-20] occurred at $20^{\circ}C$ in 50 mM Tris-Cl (pH 8) buffer with 3 M NaCl added, whereas the phase transition temperature ($T_t$) of levansucrase-ELP[V-40] was $17^{\circ}C$ with 2 M NaCl. Levansucrase was successfully purified using the phase transition characteristics of ELPs, with a recovery yield of higher than 80%, as verified by SDS-PAGE. The specific activity was measured spectrophotometrically to be 173 U/mg and 171 U/mg for levansucrase-ELP[V-20] and levansucrase-ELP[V-40], respectively, implying that the ELP-tagging system provides an efficient one-step separation method for protein purification.

Keywords

References

  1. Chaga, G. S. 2001. Twenty-five years of immobilized metal ion affinity chromatography: Past, present and future. J. Biochem. Biophys. Methods 49: 313-334 https://doi.org/10.1016/S0165-022X(01)00206-8
  2. Deo, S. K. and S. Daunert. 2001. An immunoassay for Leuenkephalin based on a C-terminal aequorin-peptide fusion. Anal. Chem. 73: 1903-1908 https://doi.org/10.1021/ac001100q
  3. Dyr, J. E. and J. Suttnar. 1997. Separation used for purification of recombinant proteins. J. Chromatogr. B 699: 383-401 https://doi.org/10.1016/S0378-4347(97)00201-6
  4. Han, Y. W. 1997. Microbial levan. Adv. Appl. Microbiol. 35: 171-194
  5. Jang, E. K., K. H. Jang, I. Koh, I. H. Kim, S. H. Kim, S. A. Kang, C. H. Kim, S. D. Ha, and S. K. Rhee. 2002. Molecular characterization of the levansucrase gene from Pseudomonas aurantiaca S-4380 and its expression in Escherichia coli. J. Microbiol. Biotechnol. 12: 603-609
  6. Jang, K. H., E. K. Jang, S.H. Kim, I. H. Kim, S. A. Kang, I. Koh, Y. I. Park, Y. J. Kim, S. D. Ha, and C. H. Kim. 2006. High-level production of low-branched levan from Pseudomonas aurantiaca S-4380 for the production of di-$\beta$-D-fructofuranose dianhydride. J. Microbiol. Biotechnol. 16: 102-108
  7. Kim, M.-J., H.-E. Park, H.-K. Sung, T. H. Park, and J. Cha. 2005. Action mechanism of transfructosylation catalyzed by Microbacterium laevaniformans levansucrase. J. Microbiol. Biotechnol. 15: 99-104
  8. Kostal, J., A. Mulchandani, and W. Chen. 2001. Tunable biopolymers for heavy metal removal. Macromolecules 34: 2257-2261 https://doi.org/10.1021/ma001973m
  9. Lee, C. Y., K. H. Kim, S. Y. Hur, J.-H. Heo, M. H. Choi, S. K. Rhee, and C. H. Kim. 2006. Enzymatic synthesis of ascorbic acid fructoside by transfructosylation using levan fructotransferase. J. Microbiol. Biotechnol. 16: 64-67
  10. Lesser, E. W. and J. A. Asenjo. 1992. Rational design of purification processes for recombinant proteins. J. Chromatogr. 584: 43-57 https://doi.org/10.1016/0378-4347(92)80008-E
  11. Lichty, J. J., J. L. Malecki, H. D. Agnew, D. J. M.-Horowitz, and S. Tan. 2005. Comparison of affinity tags for protein purification. Protein Expr. Purif. 41: 98-105 https://doi.org/10.1016/j.pep.2005.01.019
  12. McPherson, D. T., C. Morrow, D. S. Minehan, J. Wu, E. Hunter, and D. W. Urry. 1992. Production and purification of a recombinant elastomeric polypeptide, $G-(VPGVG)_{19}-VPGV$, from Escherichia coli. Biotechnol. Prog. 8: 347-352 https://doi.org/10.1021/bp00016a012
  13. McPherson, D. T., J. Xu, and D. W. Urry. 1996. Product purification by reversible phase transition following Escherichia coli expression of genes encoding up to 251 repeats of the elastomeric pentapeptide GVGVP. Protein Expr. Purif. 7: 51-57 https://doi.org/10.1006/prep.1996.0008
  14. Meyer, D. E. and A. Chilkoti. 1999. Purification of recombinant proteins by fusion with thermally responsive polypeptides. Nat. Biotechnol. 17: 1112-1115 https://doi.org/10.1038/15100
  15. Meyer, D. E., K. Trabbic-Carlson, and A. Chilkoti. 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 https://doi.org/10.1021/bp010049o
  16. Meyer, D. E. and A. Chilkoti. 2002. Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: Examples from the elastin-like polypeptide system. Biomacromolecules 3: 357-367 https://doi.org/10.1021/bm015630n
  17. O'Mullan P., M. Szakacs-Dobozi, and D. E. Eveleigh. 1991. Identification of saccharolytic enzymes of Zymomonas mobilis CP4. Biotechnol. Lett. 13: 137-142 https://doi.org/10.1007/BF01030465
  18. Roe, S. 2001. Protein Purification Applications, pp. 1-18, 2nd Ed. Oxford University Press, Great Clarendon Street, Oxford
  19. Sambrook, J. and D. W. Russell. 2001. Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
  20. Shimazu, M., A. Mulchandani, and W. Chen. 2002. Thermally triggered purification and immobilization of elastin-OPH fusions. Biotechnol. Bioeng. 81: 74-79
  21. Shuler, M. L. and F. Kargi. 2002. Bioprocess Engineering: Basic Concepts, pp. 329-384, 2nd Ed. Prentice Hall PTR, Upper Saddle River, New Jersey
  22. Smith, D. B. and K. S. Johnson. 1988. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67: 31-40 https://doi.org/10.1016/0378-1119(88)90005-4
  23. Srinivasan, U. and J. A. Bell. 1998. A convenient method for affinity purification of maltose binding protein fusions. J. Biotechnol. 62: 163-167 https://doi.org/10.1016/S0168-1656(98)00058-3
  24. Stiborova, H., J. Kostal, A. Mulchandani, and W. Chen. 2003. One-step metal-affinity purification of histidine-tagged proteins by temperature-triggered precipitation. Biotechnol. Bioeng. 82: 605-611 https://doi.org/10.1002/bit.10609
  25. Terpe, K. 2003. Overview of tag protein fusions: From molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 60: 523-533 https://doi.org/10.1007/s00253-002-1158-6
  26. Urry, D. W., T. L. Trapane, and K. U. Prasad. 1985. Phasestructure transition of the elastin polypentapeptide-water system within the framework of composition-temperature studies. Biopolymers 24: 2345-2356 https://doi.org/10.1002/bip.360241212
  27. Urry, D. W. 1997. Physical chemistry of biological free energy transductions as demonstrated by elastic proteinbased polymers. J. Phys. Chem. B 101: 11007-11028 https://doi.org/10.1021/jp972167t
  28. Urry, D. W., C. Luan, T. M. Parker, D. C. Gowda, K. U. Prasad, M. C. Reid, and A. Safavy. 1991. Temperature of polypeptide inverse temperature transition depends on mean residue hydrophobicity. J. Am. Chem. Soc. 113: 4346-4348 https://doi.org/10.1021/ja00011a057