Generation of Conformation-Specific Antibody Fragments for Crystallization of the Multidrug Resistance Transporter MdfA

Abstract

A major hurdle in membrane protein crystallography is generating crystals diffracting sufficiently for structure determination. This is often attributed not only to the difficulty of obtaining functionally active protein in mg amounts but also to the intrinsic flexibility of its multiple conformations. The cocrystallization of membrane proteins with antibody fragments has been reported as an effective approach to improve the diffraction quality of membrane protein crystals by limiting the intrinsic flexibility. Isolating suitable antibody fragments recognizing a single conformation of a native membrane protein is not a straightforward task. However, by a systematic screening approach, the time to obtain suitable antibody fragments and consequently the chance of obtaining diffracting crystals can be reduced. In this chapter, we describe a protocol for the generation of Fab fragments recognizing the native conformation of a major facilitator superfamily (MFS)-type MDR transporter MdfA from Escherichia coli. We confirmed that the use of Fab fragments was efficient for stabilization of MdfA and improvement of its crystallization properties.

The multidrug-resistance transporter MdfA fromEscherichia coli: crystallization and X-ray diffraction analysis

“…Preparation of Fab fragments. Fab fragments were generated as described previously (Jaenecke et al, 2017; Supplementary Fig. S1) according to established protocols (Day et al, 2007).…”

“…Whole IgG molecules, collected from large-scale culture supernatant of monoclonal hybridomas and purified using protein G affinity chromatography, were digested with papain (Nacalai) and Fab fragments were isolated using a Superdex 200 gelfiltration column followed by protein A affinity chromatography (Bio-Rad). This procedure resulted in the isolation of four MdfA-specific monoclonal antibodies (YN1006, YN1010, YN1074 and YN1082), the Fab fragments of each of which form a stable complex with the transporter that can be isolated using SEC (Jaenecke et al, 2017).…”

“…At pH 7.0, the melting curve of MdfA shows an apparent transition temperature T m of $58 C in the CPM assay, which is increased by $4 C in the complexes with Fabs YN1006, YN1010 and YN1082 and by $12 C in the presence of Fab YN1074 (Jaenecke et al, 2017) further analysed as a function of pH (Fig. 1b).…”

“…Prior to this study, we expressed and purified the MFS-type MDR transporter MdfA from E. coli to generate and isolate antibody Fab fragments against MdfA, with a view towards using these as potential crystallization chaperones (Hino et al, 2013). In this way, we were able to identify Fab fragments that stabilize MdfA as measured using the N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) thermostability assay (Jaenecke et al, 2017). Here, we show that the Fab fragment YN1074 is also able to suppress pH-dependent stability changes in the transporter.…”

Cryo-EM structure of the MgtE Mg2+channel pore domain in Mg2+-free conditions reveals cytoplasmic pore opening

“…Mouse monoclonal antibodies against MgtE were raised by an established method (Jaenecke et al 2018). Briefly, a proteoliposome antigen was prepared by reconstituting purified, functional MgtE at high density in phospholipid vesicles consisting of a 10:1 mixture of egg phosphatidylcholine (Avanti Polar Lipids) and the adjuvant lipid A (Sigma) to facilitate the immune response.…”