title: High yield secretion of the sweet-tasting protein lysozyme from the yeast Pichia pastoris.
abstract: Hen egg lysozyme (HEL) is one of the sweet-tasting proteins. To understand why lysozyme is sweet, the enzyme was synthesized at high yields by a recombinant method. The mature HEL gene was cloned from a Taq polymerase-amplified PCR product into the Pichia pastoris expression and secretion vector pPIC6alpha. This expression vector contains both the Saccharomyces cerevisiae pre-pro alpha-mating factor secretion signal and the blasticidin resistance gene (bsd) for selection of transformants in bacteria and yeast. Expression of HEL was carried out in fermenter cultures. Culture supernatants were concentrated by ultrafiltration and purified by CM-ion exchange chromatography. Approximately 400 mgL-1 of recombinant HEL was obtained. The high yield of recombinant lysozyme enabled us to perform a sensory analysis in humans. The purified recombinant lysozyme elicited as a sweet taste sensation as does the lysozyme purified directly from egg white, and showed full lytic activity against cells of Micrococcus luteus. These results demonstrate that the P. pastoris expression system with the blasticidin S selection system is useful in producing recombinant sweet-tasting protein in active form at a high yield.
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Masuda
authors.forename: Yuki
authors.initials: Y
authors.lastname: Ueno
authors.forename: Naofumi
authors.initials: N
authors.lastname: Kitabatake
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002645:Chickens / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D005822:Genetic Vectors; D009113:Muramidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D013649:Taste / Q000502:physiology
chemical_list: C421864:hen egg lysozyme; D009113:Muramidase