Skip to main content
SpringerLink
Log in

Column Chromatography Free Purification of Recombinant α-Amylase from Bacillus licheniformis by Tagging with Hydrophobic Elastin Like Polypeptide

  • Research Article
  • Published:
Proceedings of the National Academy of Sciences, India Section B: Biological Sciences Aims and scope Submit manuscript

Abstract

Thermal sensitive and highly hydrophobic elastin like polypeptide (ELP) tends to undergo inverse transition cycling (ITC) which can be used for non chromatographic purification. The present study reports non-chromatographic purification of industrially important α-amylase tagged with ELP and compared with IMAC (Immobilized metal affinity chromatography) purified α-amylase. amyL-Gene encoding α-amylase from Bacillus licheniformis was cloned and expressed in E. coli BL21. The expressed protein with His-tag was purified through IMAC using Ni–NTA matrix. Three ELP genes encoding repeats of pentapeptide (Val-Pro-Gly-Val-Gly)n with variable length (V = 20, 21, 22) were synthesized through PCR using overlapping primers. To generate ELP tagged α-amylase, amyL was placed at N-terminal of ELPs and transformed to E. coli BL21 for expression. After ITC, ELP22 at 30 °C showed maximum yield. α-Amylase purification through ITC and IMAC showed 2.9 and 1.72-fold purification, respectively. Furthermore, physical parameters of ELP tagged α-amylase have shown improvement with working temperature and thermal stability in comparison to His-tag α-amylase. The k cat /K m for ELP tag and His-tag α-amylase was found to be 61.4 and 23.7 mg−1 min−1, respectively, which shows that ELP-tag increased the enzyme efficiency. In conclusion, the ELP-tag purification strategy can be applied to industrially relevant enzyme for purification by the non-chromatography method.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Gupta R, Gigras P, Mohapatra H, Goswami VK, Chauhan B (2003) Microbial α-amylases: a biotechnological perspective. Process Biochem 38:1599–1616. doi:10.1016/S0032-9592(03)00053-0

    Article  CAS  Google Scholar 

  2. Benjamin S, Smitha RB, Jisha VN, Pradeep S, Sajith S, Sreedevi S, Priji P, Unni KN, Josh S (2013) A monograph on α-amylases from Bacillus spp. Adv Biosci Biotechnol 4:227–241. doi:10.4236/abb.2013.42032

    Article  CAS  Google Scholar 

  3. Tomazic SJ, Klibnov AM (1988) Mechanisms of irreversible thermal inactivation of Bacillus α-amylases. J Biol Chem 263:3092–3096

    PubMed  CAS  Google Scholar 

  4. Sassenfeld HM (1990) Engineering proteins for purification. Trends Biotechnol 4:88–93

    Article  Google Scholar 

  5. Nilsson J, Stahl S, Lundeberg J, Uhlén M, Nygren PA (1997) Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins. Protein Expr Purif 11:1–16

    Article  PubMed  CAS  Google Scholar 

  6. Urry DW (1988) Entropic elastic processes in protein mechanisms. I. Elastic structure due to an inverse temperature transition and elasticity due to internal chain dynamics. J Protein Chem 7:1–34

    Article  PubMed  CAS  Google Scholar 

  7. Urry DW (1992) Free energy transduction in polypeptides and proteins based on inverse temperature transitions. Prog Biophys Mol Biol 57:23–57. doi:10.1016/0079-6107(92)90003-O

    Article  PubMed  CAS  Google Scholar 

  8. Urry DW (1997) Physical chemistry of biological free energy transduction as demonstrated by elastic protein-based polymers. J Phys Chem 101:11007–11028. doi:10.1021/jp972167t

    Article  CAS  Google Scholar 

  9. Trabbic-Carlson K, Liu L, Kim B, Chilkoti A (2004) Expression and purification of recombinant proteins from Escherichia coli: comparison of elastin-like polypeptide fusion with an oligohistidine fusion. Protein Sci 13:3274–3284. doi:10.1110/ps.04931604

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Meyer DE, Chilkoti A (1999) Purification of recombinant proteins fusion with thermally-responsive polypeptides. Nat Biotechnol 17:1112–1115. doi:10.1038/15100

    Article  PubMed  CAS  Google Scholar 

  11. Trabbic-Carlson K, Meyer DE, Liu L, Piervincenzi R, Nath LaBean T, Chilkoti A (2004) Effect of protein fusion on the transition temperature of an environmentally responsive elastin-like polypeptide: A role for surface hydrophobicity? Protein Eng Des Sel 17:57–66. doi:10.1093/protein/gzh006

    Article  PubMed  CAS  Google Scholar 

  12. Kowalczyk T, Hnatuszko-Konka K, Gerszberg A, Kononowicz A (2014) Elastin like polypeptides as a promising family of genetically-engineered protein based polymers. World J Microbiol Biotechnol 30:2141–2152. doi:10.1007/s11274-014-1649-5

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–429

    Article  CAS  Google Scholar 

  14. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  15. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  16. Christensen T, Amiram M, Dagher S, Trabbic-Carlson K, Shamji MF, Setton LA, Chilkoti A (2009) Fusion order controls expression level and activity of elastin-like polypeptide fusion proteins. Protein Sci 18:1377–1387. doi:10.1002/pro.157

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Kim JY, Mulchandani A, Chen W (2005) Temperature-triggered purification of antibodies. Biotechnol Bioeng 90:373–379. doi:10.1002/bit.20451

    Article  PubMed  CAS  Google Scholar 

  18. Jenikova G, Lao UL, Gao D, Mulchandani A, Chen W (2007) Elastin-calmodulin scaffold for protein microarray fabrication. Langmuir 23:2277–2279

    Article  PubMed  CAS  Google Scholar 

  19. Hassouneh W, Christensen T, Chilkoti A (2010) Elastin-like polypeptides as a purification tag for recombinant proteins. Curr Protoc Protein Sci 6:11. doi:10.1002/0471140864.ps0611s61

    Article  PubMed  Google Scholar 

  20. Urry DW, Luan CH, Parker TM, Gowda DC, Prasad KU, Reid MC, Safavy A (1999) Temperature of polypeptide inverse temperature transition depends on mean residue hydrophobicity. J Am Chem Soc 113:4346–4348. doi:10.1021/ja00011a057

    Article  Google Scholar 

  21. Krishnan T, Chandra AK (1983) Purification and characterization of α-amylase from Bacillus licheniformis CUMC305. Appl Environ Microbiol 46:430–437

    PubMed  PubMed Central  CAS  Google Scholar 

  22. Moscaelli P, Boraldi F, Bochicchio B, Pepe A, Salvi AM, Quaglino D (2014) Structural characterization and biological properties of the amyloidogenic elastin-like peptide (VGGVG)3. Matrix Biol 36:15–27

    Article  CAS  Google Scholar 

  23. Urry DW, Trapane TL, Prasad KU (1985) Phase-structure transitions of the elastin polypentapeptide-water system within the framework of composition-temperature studies. Biopolymers 24:2345–2356. doi:10.1002/bip.360241212

    Article  PubMed  CAS  Google Scholar 

  24. Shimazu M, Mulchandani A, Chen W (2003) Environmentally triggered purification and immobilization of elastin-OPH fusions. Biotechnol Bioeng 81:74–79. doi:10.1002/bit.10446

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

The authors wish to thank the Department of Biotechnology (DBT) (Project No BT/PR9727/GBD/27/505/2013), New Delhi for financial support.

Author information

Authors and Affiliations

  1. Department of Biochemistry, P. D. Patel Institute of Applied Sciences, CHARUSAT, Changa, Gujarat, 388 421, India

    Rekha C. Akhani, Arti T. Patel, Manisha J. Patel, Samir R. Dedania, Jagdish S. Patel & Darshan H. Patel

  2. K. C. Patel Research and Development Centre, CHARUSAT, Changa, Gujarat, India

    Darshan H. Patel

Authors
  1. Rekha C. Akhani
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Arti T. Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Manisha J. Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Samir R. Dedania
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jagdish S. Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Darshan H. Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Darshan H. Patel.

Ethics declarations

Conflict of interest

The authors do not have any conflict of interest to declare.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Akhani, R.C., Patel, A.T., Patel, M.J. et al. Column Chromatography Free Purification of Recombinant α-Amylase from Bacillus licheniformis by Tagging with Hydrophobic Elastin Like Polypeptide. Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 88, 1249–1255 (2018). https://doi.org/10.1007/s40011-017-0862-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s40011-017-0862-z

Keywords

Search

Navigation