Cryptic Cuts of an Escort Protein

The yeast Pichia pastoris is considered to be one of the premier systems for heterologous protein expression. However, secretion and subsequent purification may be problematic. We are attempted to use E. coli Maltose Binding Protein (MBP) as an “escort” protein to improve secretion and purification of other recombinant proteins from P. pastoris. To test this, we created a fusion between human protein FKBP-12 and MBP, but found that the protein was cut between FKBP-12 and MBP. With such proteolysis preventing secretion and purification, we deleted amino acids from MBP, an IEGR region and poly-N region, which may be possible sites for proteolysis. Western blot analysis yielded results that suggest MBP proteolysis is not amino acid specific but rather depends on its three-dimensional shape