Methods to Select for Agents That Stabilize Protein Complexes

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January 28, 2016

Inventors

Jan Steyaert, Alexandre Wohlkonig, and Sarah Triest

Assignees

VIB VZW [BE/BE];Rijvisschestraat 120, B-9052 Gent (BE).
VRIJE UNIVERSITEIT BRUSSEL [BE/BE]; Pleinlaan 2, B-1050 Brussel (BE)

Abstract

The invention relates to the field of structural biology. More specifically, the invention relates to methods for the identification and characterization of biomolecular tools allowing the selective recognition and/or stabilization of distinct conformational states of protein complexes, including transient protein-protein interactions and protein-nucleic acid complexes. Such tools can be used for purification purposes, crystallization and structure determination of these stabilized protein complexes, for drug discovery, as research tools, as well as for diagnosis and treatment of diseases.

CovalX Technology Used

K100

Outcomes

In order to determine the best binding agent for stabilizing transient protein-protein interactions, a variety of cross-linkers were tested with a transient protein complex before llamas were immunized using this complex. The in vivo matured antibodies were displayed and co-selected to create nanobodies that bind to conformational epitopes of transient protein complexes.

Samples were cross-linked using the CovalX K100 reagent because of their ability to be effective over a wide range of interacting proteins in covalent complexes while exposed to physiological conditions. The antigen was diluted in antigen compatible buffer before being mixed with the cross-linked antigen and Gerbu adjuvant via up-and-down pipetting. The final mixture was injected into llamas using a standard immunization scheme.

Day	Llama Injection	Tissue Collection
Day 0	200 μg antigen + 0 μL Ag buffer + 600 μL Gerbu	10 ml pre-immune blood
Day 14	100 μg antigen + 100 μL Ag buffer + 100 μL Gerbu
Day 21	100 μg antigen + 100 μL Ag buffer + 100 μL Gerbu
Day 28	100 μg antigen + 100 μL Ag buffer + 100 μL Gerbu	10 ml immune blood
Day 35	100 μg antigen + 100 μL Ag buffer + 100 μL Gerbu
Day 42	100 μg antigen + 100 μL Ag buffer + 100 μL Gerbu
Day 45		100 ml immune blood

Using ELISA, the humoral immune response was evaluated using the blood samples collected on days 28 and 42. Further exploitation and co-selection were performed in yeast cells in order to display the nanobodies. Researchers were able to characterize the nanobody families that were present and classify them into two separate types. Thus, the method used by the researchers to select for conformation-selective binding agents is being claimed.