Effect of Its Pro-peptide Sequence on Expression of Aspergillus oryzae Alkaline Protease Gene in Pichia pastoris

GUO Ji-ping1, MA Guang2, MA Ying1 (1. College of Food Science and Engineering, Harbin Institute of Technology, Harbin 150090,China; 2.College of Life Science, Northeast Forest University, Harbin 150040, China)

Aspergillus oryzae alkaline protease gene; pro-peptide sequence; Pichia pastoris; heterologous expression

To study the essentiality of its pro-peptide sequence when Aspergillus oryzae alkaline protease (alp) gene was expressed in Pichia pastoris, the cDNA encoding mature protein of alkaline protease from A. oryzae without pro-peptide sequence was cloned into the vector pPIC9K. Then the recombinant plasmids pPIC9K/alp-m were linearized and transformed into P. pastoris GS115 competent cells. After the positive transformants were induced by methanol, the analysis of SDS-PAGE and alkaline protease activity were carried out. Besides, Northern blot was used to detect the expression of alp-m gene in transcription level. The results obtained with above methods were compared with the result obtained in our previous study where mature protein sequence of alkaline protease from A. oryzae with pro-peptide was expressed in P. pastoris. Without pro-peptide sequence, no alkaline protease was detected either in the cell or out the cell when alp gene was expressed in P. pastoris. Thus, pro-peptide sequence is required when alp gene is expressed in P. pastoris.