Soluble expression and purification of porcine pepsinogen from Pichia pastoris.
This paper presents a new system for the soluble expression and characterization of porcine pepsinogen from the methylotrophic yeast Pichia pastoris. The cDNA that encodes the zymogenic form of porcine pepsin (EC 3.4.23.1) was cloned into the EcoRI site of the vector pHIL-S1 downstream from the AOX1 alcohol oxidase promoter. After P. pastoris transformation, colonies were screened for expression of pepsinogen based on enzyme activity of the active form, pepsin. The recombinant enzyme was purified 138-fold by anion exchange and affinity column chromatography. Homogeneity was confirmed through SDS-PAGE, Western blot, and N-terminal sequencing. When compared to commercial pepsin, the recombinant pepsin had similar kinetic profiles, pH/temperature stability, and secondary/tertiary conformation. A glycosylated form was also isolated and found to exhibit kinetic and structural characteristics similar to those of the commercial and wild-type pepsin, but was slightly more thermal stable. The above results indicate that the P. pastoris expression system offers a convenient and efficient means to produce and purify a soluble form of pepsin(ogen).
Department of Food Science, University of Guelph, Guelph, Ont., Canada N1G 2W1.
Mark A
MA
Yoshimasu
Jong-Kun
JK
Ahn
Takuji
T
Tanaka
Rickey Y
RY
Yada
D000818:Animals; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D020116:Pepsinogen A / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013552:Swine / Q000235:genetics
D018076:DNA, Complementary; D011994:Recombinant Proteins; D020116:Pepsinogen A