title: Establishing the yeast Kluyveromyces lactis as an expression host for production of the saposin-like domain of the aspartic protease cirsin.
abstract: Typical plant aspartic protease zymogens comprise a characteristic and plant-specific insert (PSI). PSI domains can interact with membranes, and a role as a defensive weapon against pathogens has been proposed. However, the potential of PSIs as antimicrobial agents has not been fully investigated and explored yet due to problems in producing sufficient amounts of these domains in bacteria. Here, we report the development of an expression platform for the production of the PSI domain of cirsin in the generally regarded as safe (GRAS) yeast Kluyveromyces lactis. We successfully generated K. lactis transformants expressing and secreting significant amounts of correctly processed and glycosylated PSI, as well as its nonglycosylated mutant. A purification protocol with protein yields of ∼4.0 mg/liter was established for both wild-type and nonglycosylated PSIs, which represents the highest reported yield for a nontagged PSI domain. Subsequent bioactivity assays targeting phytopathogenic fungi indicated that the PSI of cirsin is produced in a biologically active form in K. lactis and provided clear evidence for its antifungal activity. This yeast expression system thereby emerges as a promising production platform for further exploring the biotechnological potential of these plant saposin-like proteins.
authors.affiliation: Centre for Neuroscience and Cell Biology, Coimbra, Portugal.
authors.forename: Pedro
authors.initials: P
authors.lastname: Curto
authors.forename: Daniela
authors.initials: D
authors.lastname: Lufrano
authors.forename: Cátia
authors.initials: C
authors.lastname: Pinto
authors.forename: Valéria
authors.initials: V
authors.lastname: Custódio
authors.forename: Ana Catarina
authors.initials: AC
authors.lastname: Gomes
authors.forename: Sebastián A
authors.initials: SA
authors.lastname: Trejo
authors.forename: Laura
authors.initials: L
authors.lastname: Bakás
authors.forename: Sandra
authors.initials: S
authors.lastname: Vairo-Cavalli
authors.forename: Carlos
authors.initials: C
authors.lastname: Faro
authors.forename: Isaura
authors.initials: I
authors.lastname: Simões
mesh_terms: D000935:Antifungal Agents / Q000302:isolation & purification / Q000378:metabolism; D057055:Aspartic Acid Proteases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D007716:Kluyveromyces / Q000235:genetics / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D049231:Saposins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014170:Transformation, Genetic
chemical_list: D000935:Antifungal Agents; D011994:Recombinant Proteins; D049231:Saposins; D057055:Aspartic Acid Proteases