Purification and characterization of radiolabelled biosynthetic human insulin from Escherichia coli. Kinetics of processing by antigen presenting cells.
An Escherichia coli strain transfected with a plasmid containing four linked human proinsulin genes was grown in the presence of 35S and 3H labelled amino acids to gain access to human insulin that was radiolabelled at 19 evenly distributed sites throughout the amino acid sequence. The multi-proinsulin precursor was cleaved at methionine residues with cyanogen bromide, then the individual proinsulin units were folded via their S-cysteine sulfonate derivative and converted to insulin by enzymatic digestion. Purification steps were carried out by ion-exchange and reverse-phase HPLC techniques. The final radiolabelled biosynthetic human insulin was produced at a specific activity of up to 300 Ci/mmol, and was shown to be indistinguishable from commercially available human insulin according to HPLC behavior, amino acid analysis, immunoreactivity and biological activity. A comparison of the kinetics of processing of 35S/3H-labelled biosynthetic human insulin and 125I-labelled commercial human insulin by murine TA3 hybridoma antigen presenting cells demonstrated that radiolabelled biosynthetic insulin was processed approximately 16 times slower than its iodinated counterpart. Measurable 125I TCA soluble radioactivity was detected extracellularly within 15 min whereas the same amount of extracellular TCA soluble 3H/35S radioactivity was not seen until 240 min. These results begin to address the importance of using a biosynthetically labelled protein as opposed to an iodinated protein to study how an APC handles antigen in a physiological manner.
Semple
J W
JW
Banting and Best Department of Medical Research, Toronto, Ontario, Canada.
Cockle
S A
SA
Delovitch
T L
TL
D000938:Antigen-Presenting Cells / Q000276:immunology*; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D006801:Humans; D007328:Insulin / Q000096:biosynthesis / Q000302:isolation & purification*; D007700:Kinetics; D011384:Proinsulin; D011994:Recombinant Proteins / Q000302:isolation & purification*; D013462:Sulfur Radioisotopes; D014162:Transfection; D014316:Tritium
D007328:Insulin; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D014316:Tritium; D011384:Proinsulin