| Title: | "Secretion, purification and characterization of a soluble form of the yeast KEX1-encoded protein from insect-cell cultures" |
| Author(s): | Latchinian-Sadek L; Thomas DY; |
| Address: | "Eukaryotic Genetics Group, Biotechnology Research Institute, Montreal, Canada" |
| DOI: | 10.1111/j.1432-1033.1994.tb19980.x |
| ISSN/ISBN: | 0014-2956 (Print) 0014-2956 (Linking) |
| 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 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" |
| Keywords: | "Amino Acid Sequence Animals Baculoviridae/genetics Base Sequence Carboxypeptidases/*biosynthesis/isolation & purification/metabolism Cell Line Chromatography, Ion Exchange DNA Primers *Genes, Fungal Genetic Vectors Hydrogen-Ion Concentration Isoelectric F;" |
| Notes: | "MedlineLatchinian-Sadek, L Thomas, D Y eng England 1994/01/15 Eur J Biochem. 1994 Jan 15; 219(1-2):647-52. doi: 10.1111/j.1432-1033.1994.tb19980.x" |
