Production in Escherichia coli of a rat chimeric proinsulin polypeptide carrying human A and B chains and its preparative chromatography.
A pseudohuman proinsulin coding DNA sequence (MMRPI) carrying human A and B chains, was constructed via directed mutagenesis of a previously modified rat proinsulin cDNA (MRPI) and expressed as a tryptophan (Trp)LE-proinsulin fusion protein in Escherichia coli W3110. Expression of the hybrid gene was achieved by depletion of tryptophan from the medium. The heterologous fusion protein, accumulated as insoluble inclusion bodies within the cell, was obtained by differential centrifugation and then solubilized using formic acid. At the junction of the two peptides, a methionine residue allowed proinsulin to be released from the carrier protein by cyanogen bromide treatment. The sulfonated form of this proinsulin polypeptide was easily purified, at a preparative level, using ion exchange chromatography.
Olmos
J
J
Instituto de Biotecnología, Universidad Nacional Autónoma de México, Morelos.
Cruz
N
N
Sánchez
M
M
López
M
M
Balbás
P
P
Gosset
G
G
Valle
F
F
Bolivar
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D006801:Humans; D002479:Inclusion Bodies; D008969:Molecular Sequence Data; D010957:Plasmids; D011384:Proinsulin / Q000235:genetics* / Q000302:isolation & purification; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D011384:Proinsulin