ISOLATION AND STRUCTURAL CHARACTERIZATION OF RECOMBINANT HUMAN NEU DIFFERENTIATION FACTOR EXPRESSED IN ESCHERICHIA-COLI

Recombinant human neu differentiation factor produced in engineered E. coli was isolated and subject to structural characterization. The rec ombinant molecule can be prepared to apparent purity and is active in stimulating receptor tyrosine autophosphorylation in cultural cells ex pressing HER2 receptor. The 229 amino-acid polypeptide consists of eig ht cysteines, of which two cysteines near the N-terminus are disulfide -bonded to form an immunoglobulin-like domain and the remaining six cy steines at the C-terminus cross-link to form an epidermal growth facto r-like structure. Detailed chemical characterization of the recombinan t molecule by peptide mapping in conjunction with Edman sequencing and mass spectrometry reveals that the bacterially produced recombinant n eu differentiation factor preparation is properly folded and contains the correct disulfide structure. The peptide mapping procedure is also useful in identifying abnormal peptides derived from deamidation and oxidation of Asn and Met residues, respectively.