Cloning, sequence analysis and expression in Escherichia coli of the cDNA encoding a precursor of peanut agglutinin.
The cDNA coding for pre-peanut agglutinin (PNA) was isolated from a bacterial expression library. It codes for a polypeptide of 273 amino acids composed of a hydrophobic signal peptide of 23 amino acids and a mature protein of 250 amino acids. The sequence of the latter is identical to that of native PNA, determined very recently by conventional methods, except that it contains 14 additional amino acids at the C-terminus. Bacterial cells harboring a plasmid with the prePNA-cDNA, produced two PNA cross-reacting proteins: one migrated on SDS-PAGE identically with the native lectin (apparent mol. wt. 31 kDa); the other, at 35 kDa, was a beta-galactosidase pre-PNA fusion protein. The former protein possessed an N-terminal sequence identical to that of the mature, native PNA, suggesting that it was processed from the 35 kDa prePNA precursor. Only the 31 kDa protein was exported into the bacterial periplasmic space, and had the ability to bind to galactose-Sepharose. The isolated processed protein had the same hemagglutinating activity as the native lectin, when assayed with sialidase-treated human erythrocytes. Like the native lectin, it did not agglutinate the untreated cells, was not inhibited by N-acetylgalactosamine, and was inhibited by Gal beta 1----3GalNAc 30-times more strongly than by galactose.
Rodriguez-Arango
E
E
Department of Membrane Research and Biophysics, Weizman Institute of Science, Rehovot, Israel.
Arango
R
R
Adar
R
R
Galili
G
G
Sharon
N
N
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D037102:Lectins / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D019887:Peanut Agglutinin; D011498:Protein Precursors / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D004269:DNA, Bacterial; D037102:Lectins; D019887:Peanut Agglutinin; D011498:Protein Precursors; D011994:Recombinant Proteins; C076006:peanut agglutinin precursor