Considerations in production of the prokaryotic ZIP family transporters for structural and functional studies.

Abstract

Zinc ions play essential roles as components of enzymes and many other important biomolecules, and are associated with numerous diseases. The uptake of Zn2+ and other metal ions require a widely distributed transporter protein family called Zrt/Irt-like Proteins (ZIP family), the majority members of which tend to have eight transmembrane helices with both N- and C- termini located on the extracellular or periplasmic side. Their small sizes and dynamic conformations bring many difficulties in their production for structural studies either by crystallography or Cryo-EM. Here, we summarize the problems that may encounter at the various steps of processing the ZIP proteins from gene to structural and functional studies, and provide some solutions and examples from our and other labs for the cloning, expression, purification, stability screening, metal ion transport assays and structural studies of prokaryotic ZIP family transporters using Escherichia coli as a heterologous host.