Secretion, purification and characterization of a soluble form of the yeast KEX1-encoded protein from insect-cell cultures.
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 alpha-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 (KEX1 delta) has been expressed in the baculovirus/insect cell system. The protein (Kex1 delta p) is efficiently secreted into the culture medium and was purified to apparent homogeneity with a yield of approximately 4 mg/l culture. Kex1 delta p 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 microM and Vmax 282 mumol.min-1 x mg protein-1. Insect-cell-derived Kex1 delta p processes alpha-factor-Lys-Arg, a known natural substrate, to mature active alpha-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.
Latchinian-Sadek
L
L
Eukaryotic Genetics Group, Biotechnology Research Institute, Montreal, Canada.
Thomas
D Y
DY
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002268:Carboxypeptidases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D005800:Genes, Fungal; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity; D014162:Transfection
D017931:DNA Primers; D011994:Recombinant Proteins; D002268:Carboxypeptidases; C078874:carboxypeptidase D