Cloning, expression, and characterization of the Fab fragment of the anti-lysozyme antibody HyHEL-5 Jamie A. Wibbenmeyer a , K. Asish Xavier 1 ; b , Sandra J. Smith-Gill c , Richard C. Willson a;b; * a Department of Biology and Biochemistry, University of Houston, 4800 Calhoun Ave., Houston, TX 77024-4792, USA b Department of Chemical Engineering, University of Houston, 4800 Calhoun Ave., Houston, TX 77024-4792, USA c Laboratory of Genetics, NCI, NIH, Bethesda, MD 20892, USA Received 15 October 1998; received in revised form 16 December 1998; accepted 17 December 1998 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. ß 1999 Published by Elsevier Science B.V. All rights reserved. Keywords : Antibody; Calorimetry; Fab; Lysozyme; Expression 1. Introduction The complex of the proteolytically derived Fab fragment of the anti-lysozyme antibody HyHEL-5 with hen egg lysozyme was one of the ¢rst anti- body^protein complexes to be characterized by X-ray crystallography [1], and has been the subject of numerous experimental [2^5] and computational studies [6^8]. In this paper, we report the cloning and expression in Escherichia coli of the individual chains of the Fab fragment of HyHEL-5 and their co-refolding to yield functional Fab. The resulting recombinant HyHEL-5 Fab has been characterized through MALDI-TOF mass spectrometry, N-termi- nal amino acid sequencing, equilibrium sedimenta- tion, and isothermal titration calorimetry. 2. Materials and methods 2.1. General techniques DNA manipulations were performed according to standard methodology [9]. Restriction enzymes were purchased from Promega or New England Biolabs. Transformation-competent E. coli JM 109 cells were purchased from Promega; E. coli BL-21(DE3) cells 0167-4838 / 99 / $ ^ see front matter ß 1999 Published by Elsevier Science B.V. All rights reserved. PII:S0167-4838(98)00285-4 * Corresponding author. Fax: +1-713-743-4300; E-mail : willson@uh.edu 1 Present address: Department of Chemistry, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA. Biochimica et Biophysica Acta 1430 (1999) 191^202