Introduction Pichia pastoris is a methylotrophic yeast that has been genetically engineered to express heterologous. In recent 20 years, over 700 proteins from bacteria to humans have been produced in this yeast. MBP (maltose binding protein) has been utilized as a translational fusion partner to improve the expression of foreign proteins made in E. coli. We initially explored whether MBP would serve as an expression enhancer and purification tag in Pichia pastoris, a popular eukaryotic host for heterologous protein expression. Methods SDS-PAGE and Western analysis were applied to analyze the protein expression. The secreted fusion proteins were purified by the amylose resin, digested by trypsin or endoproteinase Asp-N, and subjected to mass spectrometric analysis. Preliminary results When MBP was fused as an N-terminal partner to several cargo proteins (the two proteins were separated by a Factor Xa protease site) expressed in this yeast, proteolysis occurred between the two peptides and only MBP reached the extracellular region, which suggested that the fusion protein had been proteolyzed between MBP and cargo proteins. Furthermore, western analysis indicated the fusion proteins had been cleaved inside the yeast. Mass spectrometry analysis of MBP-FXa-FKBP12 demonstrated the Cterminus of that fusion protein was IEGR, the FXa sequence. Extensive mutagenesis of this spacer region between MBP and FKBP12 could not inhibit the cleavage. Mass spectrometric data indicated different C-termini in these mutant proteins, suggesting that different cleavage sites were used in the MBP fusions. These results provide new insights into the role of proteases in this expression system.