title: Improving expression and solubility of rice proteins produced as fusion proteins in Escherichia coli.
abstract: For proteins of higher eukaryotes, such as plants, which have large genomes, recombinant protein expression and purification are often difficult. Expression levels tend to be low and the expressed proteins tend to misfold and aggregate. We tested seven different expression vectors in Escherichia coli for rapid subcloning of rice genes and for protein expression and solubility levels. Each expressed gene product has an N-terminal fusion protein and/or tag, and an engineered protease site upstream of the mature rice protein. Several different fusion proteins/tags and protease sites were tested. We found that the fusion proteins and the protease sites have significant and varying effects on expression and solubility levels. The expression vector with the most favorable characteristics is pDEST-trx. The vector, which is a modified version of the commercially available expression vector, pET-32a, contains an N-terminal thioredoxin fusion protein and a hexahistidine tag, and is adapted to the Gateway expression system. However, addition of an engineered protease site could drastically change the expression and solubility properties. We selected 135 genes corresponding to potentially interesting rice proteins, transferred the genes from cDNAs to expression vectors, and engineered in suitable protease sites N-terminal to the mature proteins. Of 135 genes, 131 (97.0%) could be expressed and 72 (53.3%) were soluble when the fusion proteins/tags were present. Thirty-eight mature-length rice proteins and domains (28.1%) are suitable for NMR solution structure studies and/or X-ray crystallography. Our expression systems are useful for the production of soluble plant proteins in E. coli to be used for structural genomics studies.
authors.affiliation: Biochemistry Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Tsunoda
authors.forename: Nobuya
authors.initials: N
authors.lastname: Sakai
authors.forename: Koji
authors.initials: K
authors.lastname: Kikuchi
authors.forename: Shizue
authors.initials: S
authors.lastname: Katoh
authors.forename: Kayo
authors.initials: K
authors.lastname: Akagi
authors.forename: Jun
authors.initials: J
authors.lastname: Miura-Ohnuma
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Tashiro
authors.forename: Katsuyoshi
authors.initials: K
authors.lastname: Murata
authors.forename: Naoto
authors.initials: N
authors.lastname: Shibuya
authors.forename: Etsuko
authors.initials: E
authors.lastname: Katoh
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D012275:Oryza / Q000737:chemistry / Q000235:genetics*; D010940:Plant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D012995:Solubility
chemical_list: D010940:Plant Proteins; D011993:Recombinant Fusion Proteins