Part:BBa_K2601004

HOTag3 (Homo-Oligomeric Tag3)

Introduction

Background: Some membrane-less organelles, such as stress granules and P bodies, have been discovered in recent years. Proteins condense into droplets and assemble these organelles through a process called phase separation. Physically, phase separation is the transformation of a thermodynamic system from one phase to another, much like how oil and water demix from each other. According to thermodynamics, material will diffuse down the gradient of chemical potential instead of concentration. This is exactly why proteins will self organize into granules, diffusing from regions of low concentration to regions of high concentration. Here is an illustration of phase separation in cells.

Design

When we want to rationally design such an organelle and use it as a platform to achieve multi-functions, some design principles have to be followed. Interaction can bind the parts together while multivalence can make larger assemblies. In order to drive protein phase separation and formation of protein droplets, we need a multivalent module and a protein-protein interaction module. HOTag is a biopart that we use to introduce multivalence. In natural process, such as phase separation occurred during T cell signal transduction, multivalency depends on multiple repeats protein domains. But it is not ideal to use multiple repeat domains in our design, because it will not only make the scaffold extremely large but also be problematic for molecular cloning and making transgenic yeasts. Thus, instead of using multiple repeats, we turned to de novo-designed homo-oligomeric short peptides chosen by professor Xiaokun Shu’ s lab this year. These short peptides are called HO-Tag (homo-oligomeric tag).

Properties

HOTags contain approximately 30 amino acids. HOTag3 has high stoichiometry, forming hexamer spontaneously. The hexameric HOTag3, together with another tetrameric HOTag (HOTag6), can robustly drive protein phase separation upon protein interaction (achieved by the protein-protein interaction module). Thus, HOTag3/6 pair is a useful tool to investigate protein phase separation and design a synthetic organelle.

Figure

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]