Featured Technology Archive

Chaperone-Assisted Biology: Versatile Approaches by Two PSI:Biology Partnerships

2LV9 Two PSI:Biology High-Throughput-Enabled Structural Biology Partnerships are developing three different types of antibody-related chaperones to assist in various aspects of their structure and function studies. Nucleocytoplasmic Transport: A Target For Cellular Control (NPCXtals), is using next-generation sequencing and mass spectrometry to identify recombinant nanobodies, while the Chaperone-Enabled Studies of Epigenetic Regulation Enzymes (CEBS) is employing synthetic affinity binders (sABs) and small Fab-binding co-chaperones to assist in crystallization and cell biology assays. Both Partnerships are interested in establishing high-throughput methods to identify and produce these molecules.

NPCXtals is led by Michael Rout (The Rockefeller University), John Aitchison (Institute for Systems Biology), Yuh Min Chook (University of Texas Southwestern Medical Center), 4IFQ and Andrej Sali (University of California-San Francisco) and is collaborating with the NYSGRC, a PSI:Biology Center for High-Throughput Structure Determination, to establish high-throughput methods for producing larger quantities of nucleoporins (nuclear pore complex proteins) and karyopherins (nucleocytoplasmic 4JLQ transporters). This Partnership is concerned with delineating the mechanistic roles of these proteins in the process of nuclear transport and establishing their importance as potential drug targets. Since these molecules function in complex with other proteins, NPCXtals scientists are developing recombinant nanobodies, single chain antibodies from camelid species ~15kDa in size, for isolating these protein complexes. They have established a large-scale, high-affinity single-chain nanobody pipeline and as a proof-of-principle, used it to isolate nanobodies to green fluorescent protein (GFP). Two llamas were inoculated with GFP and a library of approximately 800,000 variable region sequences (VHH) was constructed using next-generation sequencing methods from their lymphocyte RNA. Screening this library yielded 150 unique full-length VHH sequences identified using mass spectroscopy in collaboration with Dr. Brian T. Chait, Camille and Henry Dreyfus Professor at The Rockefeller University. Twenty-six nanobodies with high affinity for binding GFP have been determined and structural studies of a subset are underway. These studies will provide useful information about the variety of binding interfaces generated during this immune response. Due to their high affinity and small size, nanobodies are useful for surface immobilization for affinity isolations and immunofluorescence microscopy, so these molecules will be used in future assays to bind GFP-tagged proteins and protein complexes and to aid in structural studies.

Two different reagents, one synthetic and one recombinant, are being employed by CEBS, headed by Principal Investigator Anthony A. Kossiakoff, Otho S. A. Sprague Professor in the Department of Biochemistry and Molecular Biology at the University of Chicago, in collaboration with colleagues in the US, Canada, and England. The synthetic antibody (sAB) chaperones, are “evolved” by phage display using a novel “reduced genetic code” library with a diversity of 1011 variants. The library content is strongly biased toward 2LM1 a subset of amino acid types that have been shown to be important in antibody-antigen interaction sites. Its power is derived from the fact that many more positions in the antigen recognition domains of antibodies can be diversified than in traditional phage-display libraries, without loss of antibody binding function. These sABs share a common framework based on a Fab fragment that has been engineered for stability and used in phage display. An automated pipeline for the high-throughput production of sABs using phage display techniques has been established by CEBS researchers and is described in detail by Paduch et al., 2012. These versatile molecules can be used for crystallization and phase determination, as well as custom affinity agents for in vivo co-localization. To date, several hundred distinct sABs to about 80 epigenetic protein targets have been generated, produced, and validated. sABs have been distributed to more than a dozen collaborators to study their performance in various cell-based applications.

In addition to developing automated, high-throughput methods for the production of sABs, CEBS has produced an improved Fab-binding co-chaperone based on a domain from bacterial Protein G. It is predicted that this 56-residue Fab-binder would change the shape and electrostatic properties of the Fab chaperone. This domain could be further engineered to change the surface properties of this molecule, introducing more versatility and variability. In order to increase its affinity for the heavy chain variable region of the Fab fragment, the co-chaperone was exposed to four rounds of phage display selection and resulting clones have been validated by surface plasmon resonance showing increased affinities of up to 80-fold compared to the wild-type protein domain.

Using both of these tools, CEBS will study the catalysis and regulation of histone modification enzymes. PSI:Biology Center for High-Throughput Structure Determination, NESG, will be partnering with CEBS to explore chaperone-enabled crystallization of epigenetic regulating factors. These two groups are planning a large-scale study to determine the optimal number of sABs per epigenetic protein target to guarantee successful crystallization trials. The results of this study, which will encompass many proteins of varying size, stability, and co-factor requirements, will be of particular interest to the wider structural biology community.

To explore the partnerships and collaborations of these and other PSI:Biology Centers, visit the SBKB’s new interactive network tool at http://sbkb.org/discoveries/network.html.