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 |
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Authors: | N V Sergeev N S Glukhova I V Nazimov V A Gulyaev I A Donetskii A I Miroshnikov |
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Institution: | (1) Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117871 GSP-7 Moscow, Russia |
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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.
For communication I, see 1]. |
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Keywords: | analytical biotechnology fusion protein analysis MALDI-TOF mass spectrometry peptide mapping recombinant proteins analysis reversed-phase HPLC |
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