Pichia pastoris is widely used for heterologous protein expression. Over 700 proteins from bacterial to human sources have been produced in this yeast. However, some foreign proteins, which are engineered to be secreted, are retained inside the cell. Secondly, more fusion peptides are needed to assist in the purification of proteins made in P. pastoris. Our objective is to use E.coli Maltose Binding Protein (MBP) as an “escort” protein to improve secretion and purification of recombinant proteins from P. pastoris. To determine if MBP can be used as a temporary “escort” protein for secretion and purification, a series of fusion proteins were prepared in P. pastoris. MBP was fused at the Nterminus to human FKBP12, a protein that is difficult to produce in sufficient quantity from P. pastoris. However, western analysis indicated that only the MBP portion was secreted efficiently into the culture medium, which suggested that the fusion protein had been proteolyzed between MBP and FKBP12. We hypothesized that an unknown protease inside the cell had cleaved the fusion protein at an unspecific site between the two peptides, perhaps at the Factor Xa sequence (IEGR). We created several mutations in the IEGR region and utilized mass spectrometry to identify the precise amino acids that had been cleaved. Mass spectrometry analysis with LTQ XL indicated that in the original fusion peptide, the MBP was proteolyzed after the R in the Factor Xa sequence. We used site-directed mutagenesis to create three other MBP-FKBP12 fusion proteins with modifications in or around the IEGR sequence. We are in the process of expressing new MBPFKBP12 mutants and using mass spectrometry to determine the sequence of their C-termini. Our goal is to determine the protease cleavage sites and, in the future, identify the responsible protease within the yeast secretory network.