Synthesis, cloning and expression in Escherichia coli of artificial genes coding for biologically active elongated precursors of the vasoactive intestinal polypeptide.
Synthetic genes coding for elongated precursors of the vasoactive intestinal polypeptide (VIP) were synthesized and cloned in a highly efficient Escherichia coli expression vector. The synthetic genes code for VIP with its methionine (at position 17) replaced by leucine and elongated at the C-terminus by Gly (vasoactive intestinal polypeptide-Gly, i.e. VIPa) or by Gly-Lys-Arg (vasoactive intestinal polypeptide-Gly-Lys-Arg, i.e. VIPb). The synthetic genes fused to the N-terminal part of the E. coli beta-galactosidase gene were expressed to yield high amounts of fusion proteins reaching upon induction at least 60% of the total cellular protein. The fusion proteins of 314 and 316 amino acids carrying in their C-terminal portion either the 29 or 31 amino acids long VIP precursor polypeptide were shown to be immunoreactive with VIP antisera and were further purified and cleaved by CNBr. The resulting purified peptide precursors (VIPa and VIPb) were recognized by VIP receptors in rat liver plasma membranes and by antibodies to porcine VIP in a radioimmunoassay. Both precursors activated adenylate cyclase in rat liver membranes and stimulated pancreatic secretion in the cat. The affinity and potency of the cloned precursors is close to that of VIP purified from porcine intestine, suggesting that the elongated VIP precursors may act even without being converted into the C-terminal amide form of the peptide. The elongated VIP precursors expressed in E. coli may provide a cheap, large-scale source of experimental material for studies on VIP actions.
Simoncsits
A
A
Institute of Genetics, Hungarian Academy of Sciences.
Tjörnhammar
M L
ML
Kalman
M
M
Cserpan
I
I
Gafvelin
G
G
Bartfai
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000378:metabolism; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010451:Peptide PHI / Q000096:biosynthesis* / Q000235:genetics; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D014660:Vasoactive Intestinal Peptide / Q000096:biosynthesis* / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics / Q000302:isolation & purification
D010451:Peptide PHI; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D014660:Vasoactive Intestinal Peptide; C050933:vasoactive intestinal peptide precursor; D001616:beta-Galactosidase