Recombinant rhizopuspepsinogen. Expression, purification, and activation properties of recombinant rhizopuspepsinogens.

Z Chen, G Koelsch, H P Han, X J Wang, X L Lin, J A Hartsuck, J Tang

The Journal of biological chemistry 1991 Jun 25

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A cDNA clone, which contained the complete rhizopuspepsin structure and the putative proregion, was placed in three different Escherichia coli expression vectors for the synthesis of rhizopuspepsinogen (Rpg). Recombinant Rpgs which were expressed in the cytosol of E. coli as inclusion bodies (cRpg and tRpg) were not active. After solubilization in 6 M urea and refolding by rapid dilution, both of these Rpgs were purified to homogeneity. The third zymogen, pRpg, which was secreted to the periplasmic space of E. coli with an omp leader, was fully active and also was purified. The expression level of pRpg was higher (over 40 mg/liter culture) than that of cRpg (about 1.5 mg/liter culture). Amino-terminal sequence analysis of the zymogens revealed that cRpg and pRpg contain 40 and 51 residues of prosequence, respectively. tRpg, which was expressed under the control of T7 promoter, was synthesized at 500 mg/liter culture and was purified at 50 mg/liter culture. This zymogen contained, in addition to 51 residues of proregion, 16 residues inherited from the expression vector construction. All of these Rpgs spontaneously converted to rhizopuspepsin in solutions of pH less than 5. Each of the conversions was associated with a change of molecular weight as monitored in sodium dodecyl sulfate-polyacrylamide electrophoresis. At least one intermediate of conversion was observed in the pH range of 2 to 3 for both the cRpg and pRpg zymogens. For pRpg and tRpg, kinetic data demonstrated that the Rpg to rhizopuspepsin conversion was accomplished by a first order, unimolecular reaction at pH 2. The first order kinetic constants in this pH at 15 degrees C were 1.1 and 2.4 min-1 for pRpg and tRpg, respectively. The activation rate decreased as pH was raised above pH 2. At pH greater than 3.0, rhizopuspepsin-catalyzed, second-order activation also takes place. Consequently, the recombinant Rpgs are activated by either of two cleavage mechanisms as is the case for pepsinogen. These results also support the hypothesis that Rpg is synthesized in Rhizopus chinensis as a zymogen. Rpg in the host fungus is probably activated by an acid environment of pH less than 5 in the secretory granules to become rhizopuspepsin before secretion.

Citation

Z Chen, G Koelsch, H P Han, X J Wang, X L Lin, J A Hartsuck, J Tang. Recombinant rhizopuspepsinogen. Expression, purification, and activation properties of recombinant rhizopuspepsinogens. The Journal of biological chemistry. 1991 Jun 25;266(18):11718-25