2AXON Neuroscience R&D Services SE, Bratislava, Slovakia
In Alzheimer disease and other tauopathies, tau protein is the constituent of neurofibrillary tangles [1]. Ultrastructurally, tau inclusions are made of paired helical filaments (PHFs) and straight filaments [2]. Protein tau emerges as a promising target for developing disease-modifying drugs [3]. Precise atomic-level structural information of the tau fibrils has been recently elucidated using cryo electron microscopy [4], but there are remaining unresolved questions. Structural study of complexes between tau protein fragments derived from the PHF core [5] and monoclonal antibodies specific for a conformation of tau in PHF core may help to elucidate missing parts of assembled tau in tauopathies.
Tau protein alone is not forming crystals, but using binding partners, e.g., monoclonal antibodies, it may be possible to stabilize distinct (in situ) fold of recombinant tau and likely crystallize the complex [6]. Fabs of monoclonal antibodies can thus serve as surrogate tau protein binding partners to aid tau crystallization. Fabs are traditionally prepared by papain digestion of intact antibody; however, these Fabs may be heterogeneous and may contain papain, which can cleave sensitive tau molecule during co-crystallization. Alternatively, recombinantly-expressed Fabs can be used. Recombinant Fabs are homogeneous and pure and therefore more suitable for crystallization. Expression of such recombinant antibodies requires the knowledge of the antibody sequence and a suitable cloning vector.
Aim of this work was to create a new expression vector yielding a high expression of recombinant Fab in CHO cells. The vector should have a suitable antibiotic resistance and contain restriction sites for facilitating cloning. We constructed vector pHu_7 derived from pCMV-BD vector (Stratagene), expressing recombinant proteins under the CMV promoter with a human immunoglobulin kappa light chain signal sequence allowing for secretion of the protein. Subsequently, we have inserted light and heavy chain of DC8E8, MN423 and DC25 antibody Fab [6,7]. We have verified the expression of the antibodies from the constructed vectors by a small-scale pilot experiment in CHO cells.
Summarizing, we successfully constructed expression vectors carrying the light and heavy chains of several monoclonal antibody Fabs. In the next steps we will purify Fab fragments on a large scale, characterize their quality for crystallographic experiments and co-crystallize them with selected variants of tau protein.