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Efficient solubilization, purification of recombinant extracellular α-amylase from pyrococcus furiosus expressed as inclusion bodies in Escherichia coli

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Journal of Industrial Microbiology & Biotechnology

Abstract

The gene encoding the Pyrococcus furiosus extracellular α-amylase (PFA) was amplified by PCR from P. furiosus genomic DNA and was highly expressed in Escherichia coli BL21-Codon Plus (DE3)-RIL. The recombinant α-amylase was mainly expressed in the form of insoluble inclusion bodies. An improved purification method was established in this paper. The solubilization of the inclusion bodies was achieved by 90°C treatment for 3 min in Britton–Robinson buffer at pH 10.5. The solubilized PFA was then diluted and subsequently purified by Phenyl Sepharose chromatography. The overall yield of the new purification method was about 58,000 U/g wet cells, which is higher than previously reported.

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References

  1. Bernfeld P (1955) Amylases α- and β-. Methods Enzymol 1:149–158

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Crabb WD, Mitchinson C (1997) Enzymes involved in the processing of starches to sugars. Trends Biotechnol 15:349–352

    Article  CAS  Google Scholar 

  4. Clark ED (2001) Protein refolding for industrial processes. Curr Opin Biotechnol 12:202–207

    Article  CAS  Google Scholar 

  5. Dong G, Vieille C, Savchenko A, Zeikus JG (1997a) Cloning, sequencing, and expression of the gene encoding extracellular α-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme. Appl Environ Microbiol 63:3569–3576

    CAS  Google Scholar 

  6. Dong G, Vieille C, Savchenko A, Zeikus JG (1997b) Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme. Appl Environ Microbiol 63:3577–3584

    CAS  Google Scholar 

  7. Grzybowska B, Szweda P, Synowiecki J (2004) Cloning of the thermostable α-amylase gene from Pyrococcus woesei in Escherichia coli. Mol Biotechnol 26:101–109

    Article  CAS  Google Scholar 

  8. Jørgensen S, Vorgias CE, Antranikian G (1997) Cloning, sequencing, characterization, and expression of an extracellular a-amylase from the hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli and Bacillus subtilis. J Biol Chem 268:16335–16342

    Article  Google Scholar 

  9. Khan RH, AppaRao KBC, Eshwari ANS, Totey SM, Panda AK (1998) Solubilization of recombinant ovine growth hormone with retention of native-like secondary structure and its refolding from the inclusion bodies of Escherichia coli. Biotechnol Prog 14:722–728

    Article  CAS  Google Scholar 

  10. Koch R, Spreinat A, Lemke K, Antranikian G (1991) Purification and properties of a hyperthermoactive α-amylase from archaebacterium pyrococcus woesei. Arch Microbiol 155:572–578

    Article  CAS  Google Scholar 

  11. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  12. Linden A, Niehaus F, Antranikian G (2000) Single-step purification of a recombinant thermostable a-amylase after solubilization of the enzyme from insoluble aggregates. J Chromatogr B 737:253–259

    CAS  Google Scholar 

  13. Patra AK, Mukhopadhyay R, Mukhija R, Krishnan A, Garg LC, Panda AK (2000) Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from escherichia coli. Protein Expr Purif 18:182–192

    Article  CAS  Google Scholar 

  14. Rivera MH, Lopez-Munguia, Soberon X, Saab-Rincon G (2003) alpha-Amylase from bacillus licheniformis mutants near to the catalytic site: effects on hydrolytic and transglycosylation activity. Protein Eng 16:505–514

    Article  CAS  Google Scholar 

  15. Schofield LR, Patchett ML, Parker EJ (2004) Expression, purification, and characterization of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase from Pyrococcus furiosus. Protein Expr Purif 34:17–27

    Article  CAS  Google Scholar 

  16. Singh SM, Panda AK (2005) Solubilization and refolding of bacteiral inclusion body protein. J Biosci Bioeng 99:303–310

    Article  CAS  Google Scholar 

  17. Tsumoto K, Umetsu M, Kumagai I, Ejima D, Arakawa T (2003) Solubilization of active green fluorescent protein from insoluble particles by guanidine and arginine. Biochem Biophys Res Commun 312:1383–1386

    Article  CAS  Google Scholar 

  18. Vallejo LF, Rinas U (2004) Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins. Microb Cell Fac 3:11

    Article  Google Scholar 

  19. Zeevi M, Artzi HB, Shaked H, Hadary D, Pinhassi D, Salik H, Bartfeld D (1991) Recovery of biologically active recombinant bovine growth hormone from inclusion bodies in Escherichia coli. In: White MD (ed) Biologicals from recombinant microorganisms and animal cells. Production and Recovery. VCH, Weinheim, Germany, pp 331–348

    Google Scholar 

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Acknowledgment

This work is supported by National Basic Research Program of China (973 Program) No. 2004CB719606.

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Authors and Affiliations

  1. Shanghai Institutes for Biological Sciences, Chinese Academy of Science, 500 Caobao Road, 200233, Shanghai, China

    Lisa Wang, Qinhua Zhou, Huayou Chen, Zhongmei Chu, Jianfeng Lu, Yi Zhang & Shengli Yang

  2. Graduate School of Chinese Academy of Science, 100049, Beijing, China

    Lisa Wang, Huayou Chen, Yi Zhang & Shengli Yang

  3. College of Medicine, Jiaxing University, 314001, Jiaxing, China

    Qinhua Zhou

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  1. Lisa Wang
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  2. Qinhua Zhou
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  3. Huayou Chen
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  4. Zhongmei Chu
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  5. Jianfeng Lu
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  6. Yi Zhang
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  7. Shengli Yang
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Correspondence to Yi Zhang.

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Wang, L., Zhou, Q., Chen, H. et al. Efficient solubilization, purification of recombinant extracellular α-amylase from pyrococcus furiosus expressed as inclusion bodies in Escherichia coli . J Ind Microbiol Biotechnol 34, 187–192 (2007). https://doi.org/10.1007/s10295-006-0185-1

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  • DOI: https://doi.org/10.1007/s10295-006-0185-1

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