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Purification and characterization of radiolabelled biosynthetic human insulin from Escherichia coli kinetics of processing by antigen presenting cells

Semple, John W. LU ; Cockle, Stephen A. and Delovitch, Terry L. (1988) In Molecular Immunology 25(12). p.1291-1298
Abstract

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... (More)

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 commerically 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 commerical 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 240min. 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.

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author
publishing date
type
Contribution to journal
publication status
published
subject
in
Molecular Immunology
volume
25
issue
12
pages
1291 - 1298
publisher
Pergamon
external identifiers
  • scopus:0024271497
  • pmid:3070357
ISSN
0161-5890
DOI
10.1016/0161-5890(88)90044-2
language
English
LU publication?
no
id
556c39c7-f721-4f3c-8fe8-b21c93f050a1
date added to LUP
2019-12-03 10:37:24
date last changed
2019-12-03 12:03:00
@article{556c39c7-f721-4f3c-8fe8-b21c93f050a1,
  abstract     = {<p>An Escherichia coli strain transfected with a plasmid containing four linked human proinsulin genes was grown in the presence of <sup>35</sup>S and <sup>3</sup>H 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 commerically available human insulin according to HPLC behavior, amino acid analysis, immunoreactivity and biological activity. A comparison of the kinetics of processing of <sup>35</sup>S/<sup>3</sup>H-labelled biosynthetic human insulin and <sup>125</sup>I-labelled commerical 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 <sup>125</sup>I TCA soluble radioactivity was detected extracellularly within 15 min whereas the same amount of extracellular TCA soluble <sup>3</sup>H/<sup>35</sup>S radioactivity was not seen until 240min. 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.</p>},
  author       = {Semple, John W. and Cockle, Stephen A. and Delovitch, Terry L.},
  issn         = {0161-5890},
  language     = {eng},
  month        = {01},
  number       = {12},
  pages        = {1291--1298},
  publisher    = {Pergamon},
  series       = {Molecular Immunology},
  title        = {Purification and characterization of radiolabelled biosynthetic human insulin from Escherichia coli kinetics of processing by antigen presenting cells},
  url          = {http://dx.doi.org/10.1016/0161-5890(88)90044-2},
  doi          = {10.1016/0161-5890(88)90044-2},
  volume       = {25},
  year         = {1988},
}