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Analytical biotechnology of recombinant peptides and proteins: II. A confirmation of the primary structure of fusion protein containing human proinsulin and optimization of its proteolysis by trypsin

Russian Journal of Bioorganic Chemistry volume 26, pages 461–465 (2000)Cite this article

Abstract

The kinetics of trypsin proteolysis of the fusion protein (FP) containing human proinsulin was studied by a set of analytical micromethods. These were the microcolumn reversed-phase HPLC and the qualitative identification by MALDI-TOF mass spectrometry and amino acid sequencing. The first stage of the proteolysis was shown to be the cleavage of FP into the leader fragment and proinsulin. The subsequent splitting off ofC-peptide from proinsulin results in the formation of ArgB31-ArgB32-insulin. The effect of temperature on the formation of de-ThrB30-insulin, a by-product, was also studied. The structure of FP was confirmed by the peptide mapping technique, and the leader fragment was shown to contain noN-terminal Met residue.

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Abbreviations

FP:

fusion protein containing the sequence of human proinsulin in its composition

rpHPLC:

reversed-phase HPLC

RHI:

recombinant human insulin

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Authors and Affiliations

  1. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117871, GSP-7 Moscow, Russia

    N. V. Sergeev, N. S. Glukhova, I. V. Nazimov, V. A. Gulyaev, I. A. Donetskii & A. I. Miroshnikov

Authors
  1. N. V. Sergeev
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  2. N. S. Glukhova
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  3. I. V. Nazimov
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  4. V. A. Gulyaev
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  5. I. A. Donetskii
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  6. A. I. Miroshnikov
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Corresponding author

Correspondence to I. V. Nazimov.

Additional information

For communication I, see [1].

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Sergeev, N.V., Glukhova, N.S., Nazimov, I.V. et al. Analytical biotechnology of recombinant peptides and proteins: II. A confirmation of the primary structure of fusion protein containing human proinsulin and optimization of its proteolysis by trypsin. Russ J Bioorg Chem 26, 461–465 (2000). https://doi.org/10.1007/BF02758616

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  • DOI: https://doi.org/10.1007/BF02758616

Key words

  • analytical biotechnology
  • fusion protein, analysis
  • MALDI-TOF mass spectrometry
  • peptide mapping
  • recombinant proteins, analysis
  • reversed-phase HPLC