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 recombinant molecule can be prepared to apparent purity and is active in stimulating receptor tyrosine autophosphorylation in cultural cells expressing HER2 receptor. The 229 amino-acid polypeptide consists of eight cysteines, of which two cysteines near the N-terminus are disulfide-bonded to form an immunoglobulin-like domain and the remaining six cysteines at the C-terminus cross-link to form an epidermal growth factor-like structure. Detailed chemical characterization of the recombinant molecule by peptide mapping in conjunction with Edman sequencing and mass spectrometry reveals that the bacterially produced recombinant neu 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.
Hara
S
S
Amgen, Inc., Amgen Center, Thousand Oaks, CA 91320-1789, USA.
Liu
N
N
Meng
S Y
SY
Lu
H S
HS
D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D020880:Neuregulins; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D014407:Tumor Cells, Cultured
D004220:Disulfides; D006023:Glycoproteins; D009414:Nerve Growth Factors; D020880:Neuregulins; D010455:Peptides; D011994:Recombinant Proteins; D010450:Endopeptidases