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

摘要

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 qualiative 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 of C-peptide from proinsulin results in the formation of Arg~(B31)-Arg~(B32)-insulin. The effect of temperature on the formation of de-Thr~(B30)-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 no N-terminal Met residue.