Skip to main content
Log in

Purification of alkaline phosphatase from chicken intestine by three-phase partitioning and use of phenyl-Sepharose 6B in the batch mode

  • Published:
Bioseparation

Abstract

Alkaline phosphatase from chicken intestine was purified from the crude preparation employing three-phase partitioning and by the use of phenyl Sepharose-6B in the batch mode. TPP uses a combination of ammonium sulphate and t-butanol to precipitate proteins from crude aqueous extracts. The precipitated protein forms interface between lower aqueous phase and upper organic solvent phase. The fold purification of the finally purified enzyme was 80 and the activity recovery was 61%. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of enzyme showed considerable purification and its molecular weight was found to be around 67 kDa.

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

Access this article

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

Instant access to the full article PDF.

Similar content being viewed by others

References

  • Agarwal R and Gupta MN (1995) Evaluation of glutaraldehyde-modified chitosan as a matrix for hydrophobic interaction chromatography. Anal. Chim. Acta. 313: 253-257.

    Google Scholar 

  • Basheeruddin K, Rothman V and Margolis S (1985) Immobilized E. coli alkaline phosphatase — Its properties, stability and utility in studying the dephosphorylation of proteins. Appl. Biochem. Biotechnol. 11: 133-140.

    Google Scholar 

  • 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.

    Google Scholar 

  • Chang CH and Moog F (1972) Alkaline phosphatase of the chicken duodenum I. Isolation and partial characterization of the multiple forms of duodenal phosphatase in pre-and post hatching stages. Biochim. Biophys. Acta 258: 154-165.

    Google Scholar 

  • Dennison C and Lovrein R (1997) Three-Phase Partitioning: concentration and purification of proteins. Protein Expr. Purif. 11: 149-161.

    Google Scholar 

  • Kassner AM, Lessmann M and Wasner H (1999) Degradation of the cyclic AMP antagonist prostaglandylinositol cyclic phosphate (cyclic PIP) by dephosphorylation. Biol. Chem. 380: 85-88.

    Google Scholar 

  • Kirchberger J and Kopperschlager G (1982) Preparation of homogenous alkaline phosphatase from calf intestine by dye-ligand chromatography. Prep.Biochem. 12: 29-47.

    Google Scholar 

  • Maunders MJ (1993) Alkaline Phosphatase (EC 3.1.3.1): “Enzymes of Molecular Biology”. Burrell MM, (Ed.) pp 331-341, Humana Press, New Jersey.

    Google Scholar 

  • Gupta MN and Mattiasson B (1994) Novel technologies in downstream processing. Chem. & Indus., 673-675.

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

    Google Scholar 

  • Lovrein RE, Goldensoph C, Anderson P and Odegard B (1987) Three Phase Partioning (TPP) via t-butanol: enzyme separation from crudes In: Burgess, R. (Ed.), “Protein Purification: Micro to macro.” p 131-148. Alan R. Liss, Inc., New York.

    Google Scholar 

  • Lovrien RE, Conroy MJ and Richardson TI (1995) Molecular basis for protein separations. In: Gregory, R.B., (Ed.), “Protein-solvent interactions” pp 521-553. Marcel Dekker Inc., New York

    Google Scholar 

  • Pappas A, Yang WL, Park TS and Carman GM (1998) Nucleotide-dependent tetramerization of CTP synthetase from Saccharomyces cerevisiae. J. Biol. Chem. 273: 15954-15960.

    Google Scholar 

  • Pike RN and Dennison C (1989) Protein fractionation by three-phase partitioning in aqueous/ t-butanol mixtures. Biotechnol. Bioeng. 33: 221-228.

    Google Scholar 

  • Sanden B, Eng LH and Dalhammar G (1998) An amperometric enzyme-linked immunosensor for nitrobacter. Appl. Microbiol. Biotechnol. 50: 710-716.

    Google Scholar 

  • Sharma A, Sharma S and Gupta MN (2000) Purification of wheat germ amylase by affinity precipitation. Protein Expr. Purif. 18: 111-114.

    Google Scholar 

  • Unson MD, Newton GL, Arnold KF, Davis CE and Fahey RC (1999) Improved methods for immunoassay of mycothiol. J. Clin. Microbiol. 37: 2153-2157.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sharma, A., Sharma, S. & Gupta, M. Purification of alkaline phosphatase from chicken intestine by three-phase partitioning and use of phenyl-Sepharose 6B in the batch mode. Bioseparation 9, 155–161 (2000). https://doi.org/10.1023/A:1008195729472

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1008195729472

Navigation