Human thyroid peroxidase is retained and aggregated during expression in Pichia pastoris and Saccharomyces cerevisiae.
Pavla Vasicova, Jiri Hasek, Ivana Malcova-Janatova
Lab.Cell Reproduction, Institute of Microbiology ASCR, Videnska 1083, Prague 4, 14220, Czech Republic
For expression of immunodominant region of human thyroid peroxidase (TPOidr) acting as a main antigen for human autoimmune diseases we chose a methylotrophic yeast Pichia pastoris. TPOidr was expressed from the vector pPICZalpha that enables secretion of protein via fusion with the S. cerevisiae alpha-factor prepro-peptide. Western blot analysis did not detect any TPOidr in the medium. Analysis of intracellular expression showed a dominant accumulation of TPOidr in the heavy membrane fraction. The protein appeared in the Triton X-100 soluble fraction as well as in the residual pellet. Blue-native electrophoresis revealed presence of TPOidr aggregates in the cells. Visualization of TPOidr by indirect immunofluorescence displayed the cellular sites of TPOidr accumulation. We analyzed expression and secretion of the fusion protein alpha prepro-peptide-TPOidr in S. cerevisiae. TPOidr was not secreted into medium. In the cells, the fusion protein was only present associated with membraneous structures. In summary, TPOidr was not secreted and remained stuck up with membraneous structures inside of the cells in both tested organisms. This result indicates that insufficient secretion in P. pastoris is not caused by presence of the heterologous leader sequence. More likely, it seems to be a feature of TPOidr protein that is not correctly folded and therefore has tendency to aggregate. This work was supported by the grants LN00B030 and AV0Z50200510.