title: Cloning, expression, and purification of the N-terminal domain of the Flo1 flocculation protein from Saccharomyces cerevisiae in Pichia pastoris.
abstract: Saccharomyces cerevisiae flocculation is governed by FLO genes, encoding Flo proteins (flocculins). Flo proteins are cell wall proteins consisting of three domains, sticking out of the cell wall and interacting with other yeast cells using their N-terminal mannose-binding domain. Until recently, flocculation research was focused on the genetic and cellular level. To extend the knowledge about flocculation to the protein level, we isolated the N-terminal domain of the Flo1p (N-Flo1p) that contains the mannose-binding domain, which is responsible for the strong interaction (flocculation) of S. cerevisiae cells. To obtain a high production yield and a more uniform and lower glycosylation of N-Flo1p, it was cloned in Pichia pastoris. The expression and the purification of N-Flo1p were optimised towards a one-step purification protocol. The activity of the protein, i.e. the binding of the purified protein to mannose using fluorescence spectroscopy, was demonstrated.
authors.affiliation: Lab. Structural Biology, Department of Bioengineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussel, Belgium. Katty.Goossens@vub.ac.be
authors.forename: Katty V Y
authors.initials: KV
authors.lastname: Goossens
authors.forename: Henri
authors.initials: H
authors.lastname: De Greve
authors.forename: Ronnie G
authors.initials: RG
authors.lastname: Willaert
mesh_terms: D003001:Cloning, Molecular; D005427:Flocculation; D006031:Glycosylation; D008358:Mannose / Q000378:metabolism; D037241:Mannose-Binding Lectins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D010843:Pichia; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence
chemical_list: C087578:FLO1 protein, S cerevisiae; D037241:Mannose-Binding Lectins; D029701:Saccharomyces cerevisiae Proteins; D008358:Mannose