The use of fusion proteins in cell-based assays provides a versatile way of examining important biochemical processes, such as mRNA translation, the process of synthesizing protein from an mRNA transcript.  The process of translation is regulated by multiple factors, including RNA-binding proteins.  The zinc finger-containing Zygote arrest proteins, Zar1 and Zar2, play an essential role in vertebrate development and bind mRNA. <br/>Previously, Zar1 and Zar2 fusion proteins were shown to repress mRNA translation using luciferase reporters, but this effect was modest.  Moreover, Zar1 and Zar2 fusion proteins showed low levels of protein accumulation and abundant degradation products.  In this study, it was hypothesized that the inclusion of inter-domain linkers would improve the stability of fusion proteins and their performance in translation assays.<br/>Three previously established linkers were examined, each 17 amino acids in length: one flexible linker (glycine-serine rich) and two rigid linkers (one alpha-helical and one proline rich).  The inclusion of any of the three linkers improved the stability of Zar fusion proteins, with higher levels of protein accumulation and fewer degradation products, such that protein accumulation was up to three times greater for some constructs.  Zar fusion proteins with inter-domain linkers also performed better in luciferase reporter assays, showing more robust regulation of translation, increasing translation repression from 30% to 50% with less protein required.<br/>The improved function of these proteins is allowing us to dissect the molecular mechanism of Zar-mediated translation regulation during development.  Furthermore, this study suggests the engineered junction between two domains of a fusion protein can affect the function of the domains and should be considered in protein design.

Inclusion of inter-domain linkers improves the stability and function of fusion proteins in cell-based assays

251st National Meeting (2016)

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