title: Efficient expression of SRK intracellular domain by a modeling-based protein engineering.
abstract: S-locus protein kinase (SRK) is a receptor kinase that plays a critical role in self-recognition in the Brassicaceae self-incompatibility (SI) response. SRK is activated by binding of its ligand S-locus protein 11 (SP11) and subsequently induced phosphorylation of the intracellular kinase domain. However, a detailed activation mechanism of SRK is still largely unknown because of the difficulty in stably expressing SRK recombinant proteins. Here, we performed modeling-based protein engineering of the SRK kinase domain for stable expression in Escherichia coli. The engineered SRK intracellular domain was expressed about 54-fold higher production than wild type SRK, without loss of the kinase activity, suggesting it could be useful for further biochemical and structural studies.
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
authors.forename: Kohji
authors.initials: K
authors.lastname: Murase
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Hirano
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
authors.forename: Seiji
authors.initials: S
authors.lastname: Takayama
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan. Electronic address: hakoshima@bs.naist.jp.
authors.forename: Toshio
authors.initials: T
authors.lastname: Hakoshima
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D015870:Gene Expression; D008958:Models, Molecular; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D000072417:Protein Domains; D015202:Protein Engineering; D011494:Protein Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D011494:Protein Kinases; C079832:S-receptor kinase
keywords: Protein engineering; Protein kinase; Self-incompatibility; Stable expression