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

The kinetics of trypsin proteolysis of the fusion protein (FP) containing h uman 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 t he proteolysis was shown to be the cleavage of FP into the leader fragment and proinsulin. The subsequent splitting off of C-peptide from proinsulin r esults in the formation of Arg(B31)-Arg(B32)-insulin. The effect of tempera ture on the formation of de-Thr(B30)-insulin, a by-product, was also studie d. The structure of FP was confirmed by the peptide mapping technique, and the leader fragment was shown to contain no N-terminal Met residue.