Highly efficient expression and purification system of small-size protein domains in Escherichia coli for biochemical characterization

Wen Jing Bao, Yong Guang Gao, Yong Gang Chang, Tie Ying Zhang, Xiao Jing Lin, Xian Zhong Yan, Hong Yu Hu

Research output: Contribution to journalArticlepeer-review

62 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)599-606
Number of pages8
JournalProtein Expression and Purification
Volume47
Issue number2
DOIs
StatePublished - Jun 2006

Bibliographical note

Funding Information:
This work was supported by grants from the 863 Hi-Tech program (2002BA711A13), the Chinese Academy of Sciences (KSCX1-SW-17), and the Shanghai Commission of Science and Technology (03JC14081).

Keywords

  • Biochemical characterization
  • Domain
  • Expression
  • Fusion protein
  • GB1
  • Purification

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