title: Highly efficient expression and purification system of small-size protein domains in Escherichia coli for biochemical characterization.
abstract: It is often essential to focus the study on the small-size domains of large proteins in eukaryotic cells in the post-genomic era, but the low expression level, insolubility, and instability of the domains have been continuing to hinder the massive purification of domain peptides for structural and biological investigation. In this work, a highly efficient expression and purification system based on a small-size fusion partner GB1 and histidine tag was utilized to solve these problems. Two vectors, namely pGBTNH and pGBH, were constructed to improve expression and facilitate purification. The linker and thrombin cleavage site have been optimized for minimal degradation during purification process. This system has been tested for eight domain peptides varying in size, linker, hydrophobicity, and predicted secondary structure. The results indicate that this system is achievable to produce these domain peptides with high solubility and stability for further biochemical characterization. Moreover, the fusion protein without the linker and thrombin cleavage site is also suitable for spectroscopic studies especially for NMR structural elucidation, if the target peptide is prone to precipitation or easily degraded during purification. This system will be beneficial to the research field of structure and function of small domain and peptide fragment.
authors.affiliation: Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China.
authors.forename: Wen-Jing
authors.initials: WJ
authors.lastname: Bao
authors.forename: Yong-Guang
authors.initials: YG
authors.lastname: Gao
authors.forename: Yong-Gang
authors.initials: YG
authors.lastname: Chang
authors.forename: Tie-Ying
authors.initials: TY
authors.lastname: Zhang
authors.forename: Xiao-Jing
authors.initials: XJ
authors.lastname: Lin
authors.forename: Xian-Zhong
authors.initials: XZ
authors.lastname: Yan
authors.forename: Hong-Yu
authors.initials: HY
authors.lastname: Hu
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D004926:Escherichia coli; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D011993:Recombinant Fusion Proteins; D013917:Thrombin