Recognition of Proteins by Metal Chelation-Based Fluorescent Probes in Cells

Jiang, Nan; Li, Hongyan; Sun, Hongzhe

doi:10.3389/fchem.2019.00560

Cited by 8 publications

(1 citation statement)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For instance, in vitro designed oligomeric proteins have been engineered through the interaction of His-tag with divalent cations as protein biomaterial with novel therapeutic properties 12 . Moreover, a family of metal-NTA based fluorescence probes has been developed for the intracellular visualization of His-tag proteins 13 . Immobilized metal affinity (IMA) strategy was also used to study protein-membrane interaction.…”

mentioning

confidence: 99%

Advantages and Disadvantages of His-Tagged Beta-Galactosidase

Flores

Clop

Barra

et al. 2020

Preprint

View full text Add to dashboard Cite

β-Galactosidase is one of the most important biotechnological enzyme used in the dairy industry, pharmacology and in molecular biology. In our laboratory we have overexpressed a recombinant β-galactosidase in Escherichia coli (E. coli). This enzyme differs from its native version (β-GalWT) in that 6 histidine residues have been added to the carboxyl terminus in the primary sequence (β-GalHis), which allows its purification by immobilized metal affinity chromatography (IMAC). In this work we compared the functionality and structure of both proteins and evaluated their catalytic behavior on the kinetics of lactose hydrolysis. We observed a significant reduction in the enzymatic activity of β-GalHis with respect to β-GalWT. Although, both enzymes showed a similar catalytic profile as a function of temperature, β-GalHis presented a higher resistance to the thermal inactivation and evidenced greater half-life time compared to β-GalWT. At room temperature, β-GalHis showed a fluorescence spectrum compatible with a partially unstructured protein however, it exhibited a lower tendency to the thermal-induced unfolding with respect to β-GalWT. Analytical ultracentrifugation experiments demonstrated that the population of β-GalHis molecules exhibited a higher proportion of monomers and a lower proportion of tetrameric species with respect to the His-tag free protein. The impairment of tetramerization may would explain the negative effect of the presence of His-tag on the enzymatic activity. In addition, the present results, analyzed in the context of the available literature, suggest that the effect of the His-tag is protein-specific.

show abstract

mentioning

confidence: 99%