Molecular Cloning, Expression, Purification, and Characterization of Methionine Aminopeptidase

Autor/innen

  • Srinivasulu Yeruva Jawaharlal Nehru Technological University & BiologicalE Pvt Lt
  • Muhammed Akbar Jawaharlal Nehru Technological University, Department of Biotechnology, Hyderabad, India
  • Priyanka Kola Jawaharlal Nehru Technological University, Department of Biotechnology, Hyderabad, India
  • Dindu Alekhya Jawaharlal Nehru Technological University, Department of Biotechnology, Hyderabad, India
  • Chinthala Hima Bindu Jawaharlal Nehru Technological University, Department of Biotechnology, Hyderabad, India
  • Subramanyam Mantha Green Signal Biopharma, Chennai, India

Schlagwörter:

Methionine aminopeptidase, E. coli K12, PCR, E. coli BL21 (DE3), IPTG, pET 11b, bovine serum albumin, chymotrypsinogen

Abstract

MAP enzyme can efficiently remove N-terminal methionine from recombinant proteins. In the present study, MAP gene from genomic DNA of Escherichia coli was amplified, cloned into pET 11b vector and transformed into E.coli DH5a cloning strain to confirm the insertion of MAP gene, then plasmid was transformed into E. coli BL21 (DE3) expression strain. The protein was eluted with 25 mM NaCl and showed a purity of ~ 95% which is quite acceptable for commercial applications of the enzyme. The protein was found to be active and can release N-terminal methionine from chymotrypsinogen where P1 amino acid is less bulky. MAP failed to release methionine from BSA whose P1 is a bulky and charged amino acid. The purified MAP can be used to remove methionine from proteins which have less bulky amino acids at P1 position. 

Autor/innen-Biografie

  • Srinivasulu Yeruva, Jawaharlal Nehru Technological University & BiologicalE Pvt Lt
    Department of Biotechnology

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Veröffentlicht

2012-09-04

Ausgabe

Bd. 2 Nr. 2 (2012)

Rubrik

Research Articles

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