Improving the secretion of a methyl parathion hydrolase in Pichia pastoris by modifying its N-terminal sequence

Abstract

Pichia pastoris is commonly used to express and secrete target proteins, although not all recombinant proteins can be successfully produced. In this study, we used methyl parathion hydrolase (MPH) from Ochrobactrum sp. M231 as a model to study the importance of the N-terminus of the protein for its secretion. While MPH can be efficiently expressed intracellularly in P. pastoris, it is not secreted into the extracellular environment. Three MPH mutants (N66-MPH, D10-MPH, and N9-MPH) were constructed through modification of its N-terminus, and the secretion of each by P. pastoris was improved when compared to wild-type MPH. The level of secreted D10-MPH was increased to 0.21 U/mL, while that of N9-MPH was enhanced to 0.16 U/mL. Although N66-MPH was not enzymatically active, it was secreted efficiently, and was identified by SDS-PAGE. These results demonstrate that the secretion of heterologous proteins in P. pastoris may be improved by modifying their N-terminal structures.

Figure 1. The 3D structure of MPH.

The red and green chains are chains A and B of MPH, respectively. The 10 residues at the N-terminus are represented in ball and stick form. The zinc ion is shown as a gray ball.

Figure 2. A box-whisker plot of enzyme activity in the culture supernatants of P. pastoris transformants

. Boxes denote the interquartile range (IQR) between the first and third quartiles, while the line inside the box denotes the median. Whiskers denote the lowest and highest values within 1.5-fold the IQR from the first and third quartiles, while circles denote outliers. 1 = P. pastoris clones that expressed MPH, 2 = N₆₆-MPH, 3 = D₁₀-MPH, and 4 = N₉-MPH.

Figure 3. Enzyme activity in culture supernatants.

MPH-24# (solid circle), N₆₆-MPH-5# (hollow circle), D₁₀-MPH-56# (solid triangle), and N₉-MPH-70# (hollow triangle) were induced by methanol for various time periods (as indicated on the x-axis). The MPH activities in the supernatants were determined using standard enzyme assays and are shown on the y-axis. Enzyme activity is expressed as the mean of three samples, and error bars indicate standard deviation (SD).

Figure 4. SDS-PAGE analysis of culture supernatants.

Transformants were induced by methanol as indicated, and the supernatants were analyzed by SDS-PAGE. Blots show the supernatants of the a) N₆₆-MPH-5#, b) D₁₀-MPH-56#, c) N₉-MPH-70#, and d) MPH-24# clones. Arrows indicate the positions of the bands corresponding to expressed proteins. Lane 1, standard protein molecular weight marker; lanes 2–9, culture supernatants after methanol induction for 24, 48, 60, 72, 84, 96, 108, or 120 h, respectively.

Figure 5. Analysis of mph mRNA levels.

The expression of the recombinant cDNAs was assessed by quantitative real-time PCR, using GAPDH for normalization. Data are expressed as the means of duplicate samples, and error bars indicate standard deviation (SD).