Title

Improvement Of The Crystallizability And Expression Of An Rna Crystallization Chaperone

Keywords

chaperone assisted RNA crystallography; Fab; protein engineering; shake flask expression; surface entropy reduction

Abstract

Crystallizing RNA has been an imperative and challenging task in the world of RNA research. Assistive methods such as chaperone-assisted RNA crystallography (CARC), employing monoclonal antibody fragments (Fabs) as crystallization chaperones have enabled us to obtain RNA crystal structures by forming crystal contacts and providing initial phasing information. Despite the early successes, the crystallization of large RNA-Fab complex remains a challenge in practice. The possible reason for this difficulty is that the Fab scaffold has not been optimized for crystallization in complex with RNA. Here, we have used the surface entropy reduction (SER) technique for the optimization of ΔC209 P4-P6/Fab2 model system. Protruding lysine and glutamate residues were mutated to a set of alanines or serines to construct Fab2SMA or Fab2SMS. Expression with the shake flask approach was optimized to allow large scale production for crystallization. Crystal screening shows that significantly higher crystal-forming ratio was observed for the mutant complexes. As the chosen SER residues are far away from the CDR regions of the Fab, the same set of mutations can now be directly applied to other Fabs binding to a variety of ribozymes and riboswitches to improve the crystallizability of Fab-RNA complex. The Authors 2011. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved2011 © The Authors 2011. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Publication Date

11-1-2011

Publication Title

Journal of Biochemistry

Volume

150

Issue

5

Number of Pages

535-543

Document Type

Article

Personal Identifier

scopus

DOI Link

https://doi.org/10.1093/jb/mvr093

Socpus ID

80455145019 (Scopus)

Source API URL

https://api.elsevier.com/content/abstract/scopus_id/80455145019

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