Secretion, purification and characterization of a soluble form of the yeast KEX1‐encoded protein from insect‐cell cultures

Abstract

The Saccharomyces cerevisiae KEX1 gene encodes a carboxypeptidase involved in the C‐terminal processing of the lysine and arginine residues from the precursors of K1 and K2 killer toxins and α‐factor (mating pheromone). In order to produce large quantities of this unique carboxypeptidase for structural studies, a functional soluble form was obtained by deleting 224 amino acids from the C‐terminus of the KEX1‐encoded protein which includes a putative membrane‐spanning domain. The resulting truncated KEX1 gene (KEX1Δ) has been expressed in the baculovirus/insect cell system. The protein (Kex1Δp) is efficiently secreted into the culture medium and was purified to apparent homogeneity with a yield of approximately 4 mg/l culture. Kex1Δp is a glycoprotein with a molecular mass of 56 kDa, its N‐terminal sequence is identical to that of the full‐length membrane‐associated form of the enzyme [Latchinian‐Sadek, L. & Thomas, D. Y. (1993) J. Biol. Chem. 268, 534–540], and like the full‐length enzyme it is not made as a proenzyme. For the soluble enzyme form, the optimum pH for activity was 5.5–6.0, and the apparent pI value of the protein determined by isoelectric focusing was 4.2. The enzyme cleaves arginine from the C‐terminus of the synthetic peptide benzoyl‐Phe‐Ala‐Arg with Km 335 μM and Vmax 282 μmol · min−1· mg protein−1. Insect‐cell‐derived Kex1Δp processes α‐factor‐Lys‐Arg, a known natural substrate, to mature active α‐factor in a manner similar to the membrane‐associated full‐length enzyme. This secreted form of the enzyme is a convenient source for the isolation of substantial quantities of the pure enzyme for detailed kinetic and structural studies. Copyright © 1994, Wiley Blackwell. All rights reserved