G protein-coupled receptors, like many other membrane proteins, are notoriously difficult to synthesize in conventional cellular systems. Although expression in insect cells is considered the preferred technique for structural characterizations in particular, inefficient membrane translocation, instability, toxic effects and low yields still pose clear limitations for their production in living cells. Recent studies started to explore alternative strategies for the in vitro production of problematic membrane proteins in cell-free lysates in combination with supplied membranes. We provide a detailed study on the production efficiencies and quality of G protein-coupled receptors, Fab fragments and other proteins synthesized in insect cell lysates containing endogenous microsomes. Effects of different reaction kinetics, redox conditions and sample preparations on the specific activities of synthesized proteins have been analyzed. The extent of glycosylation, membrane translocation and percentages of ligand binding active fractions of synthesized protein samples have been determined. We provide strong evidence that membrane insertion of integral membrane proteins can represent a prime limiting factor for their preparative scale in vitro production. Improved expression protocols resulting into higher production rates yielded more active protein in case of Fab fragments, but not in case of the human endothelin B receptor.
A eukaryotic cell-free system based on Spodoptera frugiperda cells was developed for the convenient synthesis of Fab antibody fragments and other disulfide bridge containing proteins. The system uses (i) a cell lysate that is mildly prepared under slightly reduced conditions, thus maintaining the activity of vesicles derived from the endoplasmic reticulum, (ii) signal peptide dependent translocation into these vesicles, and (iii) a redox potential based on reduced and oxidized glutathione. Monomeric heavy and light immunoglobulin chains are almost completely converted to highly active dimeric Fab joined by intermolecular disulfide bridges without supplementation of chaperones or protein disulfide isomerase. The applicability of the system is demonstrated by the synthesis of anti-lysozyme and anti-CD4 Fab antibody fragments yielding approximately 10 g Fab per milliliter reaction mixture. The lack of endotoxins in this system is a prerequisite that synthesized Fab can be applied directly using whole synthesis reactions in cell-based assays that are sensitive to this substance class. Moreover, the system is compatible with PCR-generated linear templates enabling automated generation of antibody fragments in a high-throughput manner, and facilitating its application for screening and validation purposes.
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Facilities for Macromolecular Crystallography at the Helmholtz-Zentrum Berlin (HZB)HZB operates three beamlines for Marcomolecular Crystallography (MX) at the electron storage ring BESSY II [1,2]. Beamlines BL14.1 and BL14.2 are dedicated to multi-wavelength anomalous dispersion (MAD) phasing methods, operating between 5.5-15.5 keV (2.25 to 0.75 Å) which cover the absorption edges of most commonly used heavy atom derivatives. State-of-the-art detector technologies, including the PILATUS 6M (BL14.1) and PILATUS3S 2M (BL14.2) detectors (Dectris, Switzerland) enable high quality and rapid data collection. BL14.1 provides a high degree of automation with an MD2 microdiffractometer equipped with a mini-kappa axis and reliable sample changing is realized by a CATS sample changing robot. This beamline also features an in situ plate screening option. On the fully refurbished beamline BL14.2 an automated sample changer G-ROB, which has a high capacity sample dewar hosting up to 294 samples, supporting both, SPINE and UNIpuck standard allows high-throughput data collection. Beamline BL14.3 is a fixed energy station operating at 13.8 keV (0.89 Å).
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