Synthesis of chimeric mouse nerve growth factor precursor and human beta-nerve growth factor in Escherichia coli: immunological properties.
The complete mouse prepro-nerve growth factor (NGF) DNA was fused to the carboxyl terminus of the beta-galactosidase (lac-z) gene of Escherichia coli. Similarly, a genomic fragment encoding the human NGF comprising codons 11 to 106 (from a total of 118) was fused to the fifth codon of the amino terminus of beta-galactosidase. Both bacterial vectors produce high amounts of the chimeric proteins. After cell lysis most of the chimeric mouse preproNGF protein is insoluble and appears in the pellet, whereas the majority of the chimeric human beta-NGF remains in the supernatant. Purification of the fusion proteins from the soluble fraction was achieved by affinity chromatography to p-aminophenyl beta-D-thio-galactoside Sepharose. Yields of the purified chimeric proteins were increased threefold to fourfold by the addition of protease inhibitors in the lysis and chromatography buffers. Their antigenic similarity to the preproNGF and mouse beta-NGF was examined by their interaction to sera raised against synthetic peptides which reproduce sequences of the precursor protein and to sera directed against native and denatured mouse beta-NGF using enzyme-linked immunoabsorbent assay (ELISA) techniques. Antibodies to the peptide N2 (-163 to -139) interacted with high affinity with the chimeric mouse preproNGF protein. Antisera to native and denatured mouse beta-NGF interacted with both chimeric proteins but with a variable degree of affinity. These results provide direct evidence that certain antisera to mouse beta-NGF can cross-react with the human beta-NGF molecule.
Dicou
E
E
INSERM U, Centre Hospitalier Régional, Angers, France.
Houlgatte
R
R
Lee
J
J
von Wilcken-Bergmann
B
B
D000818:Animals; D002678:Chimera; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009414:Nerve Growth Factors / Q000096:biosynthesis* / Q000276:immunology; D010957:Plasmids; D011498:Protein Precursors / Q000096:biosynthesis* / Q000276:immunology; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
D009414:Nerve Growth Factors; D011498:Protein Precursors