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Site-Specific Cleavage of Fusion Proteins
Adam Charlton
Summary
Where an affinity tag has served its purpose, it may become desirable to remove it
from the protein of interest. This chapter describes the removal of such fusion partners
from the intended protein product by cleavage with site-specific endoproteases. Methods
to achieve proteolytic cleavage of the fusion proteins are provided, along with techniques
for optimizing the yield of authentic product.
Key Words: Fusion protein; affinity tag; site-specific proteolysis; protease; proteolytic
cleavage.
1. Introduction
The use and benefits of affinity tags is the subject of this book; although
when the tag has served its purpose, it is often desirable to remove it to obtain
homogeneous protein product of native size and sequence. The use of sitespecific
endoproteases to facilitate this removal is an approach that has gained
considerable favour in recent times. There are many reasons for this widespread
adoption, but foremost amongst these is that site-specific proteases recognize
long, uncommon amino acid sequences that are highly unlikely to be found
within the protein of interest. Also, as proteases are themselves quite labile
proteins, sensitive to extremes of temperature or chemical environment, proteolytic
cleavage systems tend to function in mild conditions that may enhance
protein product stability. Finally, many site-specific proteases act after their
From: Methods in Molecular Biology, vol. 421: Affinity Chromatography: Methods and Protocols, Second Edition
Edited by: M. Zachariou © Humana Press, Totowa, NJ
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