Gene synthesis, expression in Escherichia coli and purification of immunoreactive human insulin-like growth factors I and II. Application of a modified HPLC separation technique for hydrophobic proteins.
We have expressed synthetic genes encoding human insulin-like growth factors I and II in large quantities in Escherichia coli as fusion proteins with the 300 N-terminal amino acids of the E. coli trpE gene product. The resulting hybrid proteins were purified from the insoluble fraction of crude bacterial lysates using a rapid HPLC separation procedure employing a C8 reversed-phase column and a gradient of 2-propanol in formic acid. With the procedure we obtained in volatile solvents highly purified fusion proteins that were used for further biochemical and immunological procedures. Here we describe biochemical characteristics of the bacterially expressed fusion proteins and demonstrate that these proteins share substantial antigenic properties with the native polypeptides allowing the establishment of highly specific monoclonal antibodies.
Hummel
M
M
Institute of Pathology, Klinikum Steglitz, Free University of Berlin.
Herbst
H
H
Stein
H
H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis* / Q000276:immunology; D007335:Insulin-Like Growth Factor II / Q000096:biosynthesis* / Q000276:immunology; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013002:Somatomedins / Q000096:biosynthesis*
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000939:Epitopes; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II