Introduction
Structural biologists often work on proteins that lack accurate domain annotations. When the full-length protein cannot be expressed and a domain-focused approach is necessary, problems arise since it is unclear how to design high yielding, soluble expression constructs.
Some proteins have little or no sequence similarity to others and this prevents domain identification using multiple sequence alignments. More often, some functional annotation exists e.g. from mutagenesis or deletion studies, but these regions do not define well the structural boundaries. Even when a soluble construct is obtained, disordered extensions may confound crystallisation attempts.
ESPRIT allows us to overcome these problems by applying a random library approach, coupled to robotically-assisted high throughput screening.
Presentation
Using the user’s knowledge of their target, the optimal strategy is defined through discussion. Construction of the library and screening takes approximately 5 weeks, possibly involving a short visit by the user to the platform to assist with experiments.
Due to the uniqueness of the approach and the significant time input of the platform lab, academic projects should be considered as collaborations. Only the direct costs of consumables and maintenance for the project will be invoiced.
A simple unacknowledged industrial service mode is also possible invoiced at a full-cost tariff.
Keywords
Soluble protein expression, domain definition, directed evolution, random library.
Contact
Platform engineer: Philippe Mas
Director: Darren Hart
Equipment
2 Kbiosystem colony picking and arraying robots
Typhoon 3 wavelength fluorescent scanner
HiGro microtitre plate incubator
Access
Academic projects can be submitted via Instruct for funding of consumable and visit costs. Alternatively, projects can be invoiced via the ISBG.
Pricing
Consumable cost are in the order of 1000-1500 € per experiment (depending on specification). Please feel free to discuss with us the specification and possible funding routes.
Website
ESPRIT platform is managed and hosted here
psb-grenoble.eu
Overview of the ESPRIT technology
Step 1) Random truncation libraries of the target gene are constructed using exonuclease III where all termini are available for testing as domain boundaries.
Step 2) Up to 28,000 constructs in E. coli are isolated into 384 well plates using colony picking robotics.
Step 3a) (During user visit) Clones are arrayed onto nitrocellulose membranes for expression analysis using in vivo biotinylation as a marker for solubility.
Step 3b) Clones expressing both N-ter 6xHis tags and C-ter biotinylated biotin acceptor peptide are identified by fluorescence imaging.
Step 4) Ninety-six positive clones are induced in liquid cultures and NiNTA affinity purified using a plate base purification to confirm the solubility phenotype.
Steps 1 & 2 are performed by the platform prior to a user visit. During an optional visit, the user may assist in steps 3 & 4.
Publications
1. Hart DJ, Waldo GS (2013) Library methods for structural biology of challenging proteins and their complexes. Curr. Opin. Struct. Biol. 23:403–408.
2. Yumerefendi H, Desravines DC, Hart DJ (2011) Library-based methods for identification of soluble expression constructs. Methods San Diego Calif 55:38–43.
3. An Y, Yumerefendi H, Mas PJ, Chesneau A, Hart DJ (2011) ORF-selector ESPRIT: A second generation library screen for soluble protein expression employing precise open reading frame selection. J. Struct. Biol. 175:189–197.
4. An Y, Meresse P, Mas PJ, Hart DJ (2011) CoESPRIT: a library-based construct screening method for identification and expression of soluble protein complexes. PloS One 6:e16261.
5. Hart DJ, Tarendeau F (2006) Combinatorial library approaches for improving soluble protein expression in Escherichia coli. Acta Crystallogr. D Biol. Crystallogr. 62:19–26.
6. Tarendeau F, Boudet J, Guilligay D, Mas PJ, Bougault CM, Boulo S, Baudin F, Ruigrok RWH, Daigle N, Ellenberg J, Cusack S, Simorre J-P, Hart DJ (2007) Structure and nuclear import function of the C-terminal domain of influenza virus polymerase PB2 subunit. Nat. Struct. Mol. Biol. 14:229–233.
7. Nadal M, Mas PJ, Blanco AG, Arnan C, Solà M, Hart DJ, Coll M (2010) Structure and inhibition of herpesvirus DNA packaging terminase nuclease domain. Proc. Natl. Acad. Sci. U. S. A. 107:16078–16083.
The ESPRIT platform is managed and located at EMBL.