Abstract
In 1988 the preceding journal of Nature Biotechnology, Bio/Technology, reported a work by Hopp and co-workers about a new tag system for the identification and purification of recombinant proteins: the FLAG-tag. Beside the extensively used hexa-his tag system the FLAG-tag has gained broad popularity due to its small size, its high solubility, the presence of an internal Enterokinase cleavage site, and the commercial availability of high-affinity anti-FLAG antibodies. Surprisingly, considering the heavy use of FLAG in numerous laboratories world-wide, we identified in insect cells a post-translational modification (PTM) that abolishes the FLAG-anti-FLAG interaction rendering this tag system ineffectual for secreted proteins. The present publication shows that the tyrosine that is part of the crucial FLAG epitope DYK is highly susceptible to sulfation, a PTM catalysed by the enzyme family of Tyrosylprotein-Sulfo-transferases (TPSTs). We showed that this modification can result in less than 20% of secreted FLAG-tagged protein being accessible for purification questioning the universal applicability of this established tag system.
Highlights
With high-throughput sequencing and ready-to-use gene synthesis becoming more and more routine for all laboratories, the focus for the efficient production of recombinant proteins has shifted towards facilitating the expression and subsequent purification of the encoded proteins
Our results clearly show that the tyrosine, that is part of the crucial FLAG epitope DYK, is highly susceptible to tyrosine sulfation, a post-translational modification (PTM) catalyzed by the enzyme family of Tyrosine-Protein-Sulfo-Transferases (TPSTs) in the trans-Golgi network
The higher expression levels of the TB-based NA-construct as well as its higher molecular weight were corroborated by gel filtration chromatography showing a four-fold higher absorption and faster elution compared to GCN-pLI-NA (Fig. 2D)
Summary
With high-throughput sequencing and ready-to-use gene synthesis becoming more and more routine for all laboratories, the focus for the efficient production of recombinant proteins has shifted towards facilitating the expression and subsequent purification of the encoded proteins. To allow efficient purification and to overcome known problems of protein production such as aggregation, inefficient translation, limited solubility, or degradation, affinity tag systems have become an indispensable tool [1]. Tags can promote proper folding, reduce aggregation, or increase solubility thereby increasing the yields of fused recombinant proteins. Beside the omnipresent hexahis tag alternative tag systems have been developed over the years all with different strengths and weaknesses. From these non-histag-systems (e.g. MBP, GST, CBP, STREP, myc, FLAG [1]) the FLAG tag is one of the most commonly used systems
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