Cloning, expression, and characterization of the Fab fragment of the anti-lysozyme antibody HyHEL-5

Jamie A. Wibbenmeyer, K. Asish Xavier, Sandra J. Smith-Gill, Richard C. Willson

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

Hybridoma cDNAs encoding the individual chains of the Fab fragment of the well characterized murine monoclonal antibody HyHEL-5 were cloned and sequenced. The recombinant Fab fragment was produced by expressing each chain in a separate Escherichia coli pET vector, denaturing inclusion bodies and co-refolding. Characterization of the purified Fab by MALDI-TOF mass spectrometry and N-terminal amino acid sequencing demonstrated proper processing of the individual chains. The association of the recombinant Fab fragment with hen egg lysozyme and the avian epitope variant bobwhite quail lysozyme was found by isothermal titration calorimetry to have energetics very similar to that of the HyHEL-5 IgG. Heterologous expression of the HyHEL-5 Fab fragment opens the way to structure/function studies in this well-known system. Copyright (C) 1999.

Original languageEnglish (US)
Pages (from-to)191-202
Number of pages12
JournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Volume1430
Issue number2
DOIs
StatePublished - Mar 19 1999

Keywords

  • Antibody
  • Calorimetry
  • Expression
  • Fab
  • Lysozyme

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Structural Biology
  • Biophysics

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