A gene fusion system for generating antibodies against short peptides.
A novel method to obtain specific antibodies against short peptides is described, involving synthesis of the corresponding oligodeoxynucleotides followed by cloning into a new set of fusion vectors, pEZZ8 and pEZZ18, based on two synthetic IgG-binding domains (ZZ) of Staphylococcus aureus protein A. The soluble gene fusion product thus obtained, can be collected from the culture medium of Escherichia coli and rapidly recovered in a one-step procedure by IgG affinity chromatography. The system was used to express a fusion protein consisting of the two Z fragments and the C-terminal part [amino acids (aa) 57-70] of human insulin-like growth factor I (IGF-I). This 16-kDa protein was purified by affinity chromatography on IgG Sepharose and antibodies were raised in rabbits. The fusion protein elicited peptide-specific antibodies, as measured by solid-phase radioimmuno assay and Western blotting, reactive with both synthetic C-terminal peptide and the native human IGF-I protein. The results suggests that the gene fusion system can be used for efficient antibody production against short peptides encoded by synthetic oligodeoxynucleotides.
Löwenadler
B
B
Kabigen AB, Stockholm, Sweden.
Jansson
B
B
Paleus
S
S
Holmgren
E
E
Nilsson
B
B
Moks
T
T
Palm
G
G
Josephson
S
S
Philipson
L
L
Uhlén
M
M
D000595:Amino Acid Sequence; D000917:Antibody Formation; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010455:Peptides / Q000235:genetics* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics*
D009838:Oligodeoxyribonucleotides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I