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• Flagellar filament

Introduction

Our plan to display flagellar filaments on the yeast surface consists of mainly three steps:

• 1. Display flagellin monomers on the yeast surface.
• 2. Put the yeasts obtained in step one in a solution with sufficiently high concentrations of flagellin monomers and certain anions (known as good “salting-outers”) to promote spontaneous nucleation on the yeast surface.
• 3. Transfer the yeasts obtained in step two, which have flagellin seeds on their surface, to a new buffer with physiological ionic strength and pH that also has a sufficiently high concentration of flagellin monomers. Under this condition, the flagellin seeds on the yeasts’ surface will elongate into flagellar filaments.

If we want to use this system to display enzymes, we only need to fuse the desired enzymes (often along the same chain of reactions) with the flagellin monomers, and then the obtained yeasts from the above steps will have a much larger number of enzymes displayed on it than ordinary yeasts.

We shall give a brief explanation of the above design. First note that while there is a lot of literature about Salmonella flagellins but very little about E. coli, for safety reasons we can only use E. coli flagellins in our design. However, since the flagellar polymerization of Salmonella and that of E. coli are very similar (Kondoh and Hotani, 1974), we can transfer some of the previous results concerning Salmonella flagellar filaments to E. coli.

Lino (1974) gave a review of the assembly of Salmonella flagellins in vitro. There are several characteristics worth noting. First, in vitro assembly is possible even when the flagellin seed is attached to a cell body, which confirms the fundamental validity of our design. Second, the assembly of flagellins in vitro has two steps, nucleation and elongation. Nucleation forms the seeds upon which elongation takes place. Under physiological ionic strength and pH, flagellin monomers can grow on seeds to form flagellar filaments, but they cannot nucleate into seeds. Spontaneous nucleation can only happen in the presence of high concentration of certain anions (e.g. $SO_4^{2-}$, $F^-$, citrate ions) and neutral pH. The second and third steps of our design correspond to the nucleation process and the elongation process respectively.

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