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The intervening removable affinity tag (iRAT) production system facilitates Fv antibody fragment‐mediated crystallography
Author(s) -
Nomura Yayoi,
Sato Yumi,
Suno Ryoji,
Horita Shoichiro,
Iwata So,
Nomura Norimichi
Publication year - 2016
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.3035
Subject(s) - crystallization , chemistry , antibody , immunoglobulin light chain , recombinant dna , biochemistry , chaperone (clinical) , protease , computational biology , biology , gene , enzyme , genetics , medicine , organic chemistry , pathology
Fv antibody fragments have been used as co‐crystallization partners in structural biology, particularly in membrane protein crystallography. However, there are inherent technical issues associated with the large‐scale production of soluble, functional Fv fragments through conventional methods in various expression systems. To circumvent these problems, we developed a new method, in which a single synthetic polyprotein consisting of a variable light (V L ) domain, an intervening removable affinity tag (iRAT), and a variable heavy (V H ) domain is expressed by a Gram‐positive bacterial secretion system. This method ensures stoichiometric expression of V L and V H from the monocistronic construct followed by proper folding and assembly of the two variable domains. The iRAT segment can be removed by a site‐specific protease during the purification process to yield tag‐free Fv fragments suitable for crystallization trials. In vitro refolding step is not required to obtain correctly folded Fv fragments. As a proof of concept, we tested the iRAT‐based production of multiple Fv fragments, including a crystallization chaperone for a mammalian membrane protein as well as FDA‐approved therapeutic antibodies. The resulting Fv fragments were functionally active and crystallized in complex with the target proteins. The iRAT system is a reliable, rapid and broadly applicable means of producing milligram quantities of Fv fragments for structural and biochemical studies.