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Protein Sci. 2019 Apr;28(4):823-836. doi: 10.1002/pro.3580. Epub 2019 Feb 4.

Application of the NZ-1 Fab as a crystallization chaperone for PA tag-inserted target proteins.

Author information

1
Graduate School of Medical Life Science, Yokohama City University, Yokohama, Japan.
2
Tohoku University Graduate School of Medicine, Tohoku, Japan.

Abstract

An antibody fragment that recognizes the tertiary structure of a target protein with high affinity can be utilized as a crystallization chaperone. Difficulties in establishing conformation-specific antibodies, however, limit the applicability of antibody fragment-assisted crystallization. Here, we attempted to establish an alternative method to promote the crystallization of target proteins using an already established anti-tag antibody. The monoclonal antibody NZ-1 recognizes the PA tag with an extremely high affinity. It was also established that the PA tag is accommodated in the antigen-binding pocket in a bent conformation, compatible with an insertion into loop regions on the target. We, therefore, explored the application of NZ-1 Fab as a crystallization chaperone that complexes with a target protein displaying a PA tag. Specifically, we inserted the PA tag into the β-hairpins of the PDZ tandem fragment of a bacterial Site-2 protease. We crystallized the PA-inserted PDZ tandem mutants with the NZ-1 Fab and solved the co-crystal structure to analyze their interaction modes. Although the initial insertion designs produced only moderate-resolution structures, eliminating the solvent-accessible space between the NZ-1 Fab and target PDZ tandem improved the diffraction qualities remarkably. Our results demonstrate that the NZ-1-PA system efficiently promotes crystallization of the target protein. The present work also suggests that β-hairpins are suitable sites for the PA insertion because the PA tag contains a Pro-Gly sequence with a propensity for a β-turn conformation.

KEYWORDS:

antigen-binding fragment; crystallization chaperone; epitope tag insertion; monoclonal antibody; protein crystallography

PMID:
30666745
PMCID:
PMC6424065
[Available on 2020-04-01]
DOI:
10.1002/pro.3580

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