Applies To Polypeptides Prepared By E. Coli Efficient Expression System

Proteins are often rather large in size and comprise many domains or modules in eukaryotic cells in the post-genomic era. Protein domains are relatively independent units in structure and function, which are the bases of the protein diversity and specificity. For this case, it is very common to isolate small-size protein domains for structural and functional studies especially for NMR structural elucidation. However, 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. For example, peptide synthesis is highly cost and relatively low in yield, especially for larger peptide (e.g>50 residues). Recombinant expression is an alternative strategy for getting the small-size domains, but it usually gets low expression. Much work in recent years has focused on expressing domain peptides in E. coli in a fusion way.In this work, a highly efficient expression and purification system, pGBH and pGBTNH based on a small-size fusion partner GB1, was utilized to solve these problems. Proteolytic cleavage site is feasible to generate natural biologically active domain peptides. The linker has been optimized for minimal degradation during purification process. C-terminal histidine tag was constructed to facilitate purification.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, this system will be beneficial to the research field of structure and function of small domain and peptide fragment, including the liquid state secondary structures of different domain peptides for CD study and the ability of isotopic labelling of peptides for NMR study. We also demonstrated the ability of this system to use a fluorometric method for protein aggregation study in vitro and to practise in general research applications.