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Fatty acid-binding protein from rat liver is shown to bind the fluorescent fatty acid probe dansyl undecanoic acid. Binding is accompanied by a shift in the fluorescence emission maximum from 550 nm to 500 nm and a 60-fold fluorescence enhancement at 500 nm. These spectral properties have allowed the use of this probe to detect and quantify microgram amounts of liver fatty acid-binding protein during purification procedures. In conjunction with h.p.l.c. the method allows the rapid estimation of liver fatty acid-binding protein in biological samples. The validity of the method is demonstrated by measuring the concentration of fatty acid-binding protein in livers from control and hypolipidaemic-drug-treated rats. The dramatic diurnal rhythm previously reported for this protein [Dempsey (1984) Curr. Top. Cell. Regul. 24, 63-86] was not observed with this method.

The critical role played by IgE in allergic asthma is well-documented and clinically precedented, but some patients in whom IgE neutralization may still offer clinical benefit are excluded from treatment with the existing anti-IgE therapy, omalizumab, due to high total IgE levels or body mass. In this study, we sought to generate a novel high affinity anti-IgE antibody (MEDI4212) with potential to treat a broad severe asthma patient population. Analysis of body mass, total and allergen-specific IgE levels in a cohort of severe asthmatics was used to support the rationale for development of a high affinity IgE-targeted antibody therapeutic. Phage display technology was used to generate a human IgG1 lead antibody, MEDI4212, which was characterized in vitro using binding, signaling and functional assay systems. Protein crystallography was used to determine the details of the interaction between MEDI4212 and IgE. MEDI4212 bound human IgE with an affinity of 1.95 pM and was shown to target critical residues in the IgE Cε3 domain critical for interaction with FcεRI. MEDI4212 potently inhibited responses through FcεRI and also prevented the binding of IgE to CD23. When used ex vivo at identical concentration, MEDI4212 depleted free-IgE from human sera to levels ~1 log lower than omalizumab. Our results thus indicate that MEDI4212 is a novel, high affinity antibody that binds specifically to IgE and prevents IgE binding to its receptors. MEDI4212 effectively depleted free-IgE from human sera ex vivo to a level (1 IU/mL) anticipated to provide optimal IgE suppression in severe asthma patients.

TGF-␤ isoforms are key modulators of a broad range of biological pathways and increasingly are exploited as therapeutic targets. Here, we describe the crystal structures of a pan-TGF-␤ neutralizing antibody, GC-1008, alone and in complex with TGF-␤3. The antibody is currently in clinical evaluation for idiopathic pulmonary fibrosis, melanoma, and renal cell cancer. GC-1008 recognizes an asymmetric binding interface across the TGF-␤ homodimer with high affinity. Whereas both cognate receptors, TGF-␤-receptor types I and II, are required to recognize all 3 TGF-␤ isoforms, GC-1008 has been engineered to bind with high affinity to TGF-␤1, 2, and 3 via a single interaction surface. Comparison with existing structures and models of TGF-␤ interaction with its receptors suggests that the antibody binds to a similar epitope to the 2 receptors together and is therefore a structurally different but functionally identical mimic of the binding mode of both receptors.cancer ͉ fibrotic diseases ͉ TGF-␤/antibody complex ͉ TGF-␤ signaling ͉ X-ray structure T he transforming growth factor ␤ (TGF-␤) superfamily of cytokines comprises Ͼ30 structurally related proteins that are involved in the regulation of a wide variety of biological processes such as cell proliferation, differentiation, and expression of extracellular matrix proteins (1, 2). Members of this family are Ϸ25-kDa homodimeric molecules with a similar structural framework in which the 2 monomers are covalently linked via a disulfide bridge (3-8). Three different isoforms of TGF-␤ are known in mammals that share a sequence identity of 70-82% (2). All 3 isoforms of TGF-␤ are expressed as latent or inactive propeptides that can be activated by a diverse number of physiologically and pathophysiologically associated mechanisms such as thrombospondin-1, integrin ␣v␤6, reactive oxygen species, and low pH (9, 10). Expression of TGF-␤ isoforms and the activation of latent TGF-␤ to mature active protein are tightly regulated in normal physiology, and a dysregulation of this process has been described in many pathological conditions leading to an enhanced activity of TGF-␤ in diseases such as fibrotic disease and some malignancies. Gene-deletion studies in vivo indicate that the 3 mammalian isoforms of TGF-␤ have nonoverlapping activities essential for vascular development and the regulation of immune cell function (11-13). However, in vitro, the biological activities of the 3 isoforms of TGF-␤ are almost identical. TGF-␤1 and TGF-␤3 trigger the cellular signaling cascade upon binding to the extracellular domains of 2 transmembrane receptors, known as TGF-␤ receptor types I and II, forming a ternary complex. This complex assembly occurs first through high affinity binding of the cytokines to their TGF-␤ receptor type II, followed by the recruitment of the TGF-␤ receptor type I (14, 15). The binding potency of TGF-␤ to its type II receptor is isoform-dependent, with the highest binding affinities for TGF-␤1 and 3 and a 10-to 20-fold-smaller binding affinity for 17). The formation ...

The expression and subsequent purification of mammalian recombinant proteins is of critical importance to many areas of biological science. To maintain the appropriate tertiary structure and post-translational modifications of such proteins, transient mammalian expression systems are often adopted. The successful utilisation of these systems is, however, not always forthcoming and some recombinant proteins prove refractory to expression in mammalian hosts. In this study we focussed on the role of different N-terminal signal peptides and residues immediately downstream, in influencing the level of secreted recombinant protein obtained from suspension HEK293 cells. Using secreted alkaline phosphatase (SEAP) as a model protein, we identified that the +1/+2 downstream residues flanking a heterologous signal peptide significantly affect secreted levels. By incorporating these findings we conducted a comparison of different signal peptide sequences and identified the most productive as secrecon, a computationally-designed sequence. Importantly, in the context of the secrecon signal peptide and SEAP, we also demonstrated a clear preference for specific amino acid residues at the +1 position (e.g. alanine), and a detrimental effect of others (cysteine, proline, tyrosine and glutamine). When proteins that naturally contain these “undesirable” residues at the +1 position were expressed with their native signal peptide, the heterologous secrecon signal peptide, or secrecon with an additional alanine at the +1 or +1 and +2 position, the level of expression differed significantly and in an unpredictable manner. For each protein, however, at least one of the panel of signal peptide/adjacent amino acid combinations enabled successful recombinant expression. In this study, we highlight the important interplay between a signal peptide and its adjacent amino acids in enabling protein expression, and we describe a strategy that could enable recombinant proteins that have so far proved refractory to expression in HEK293 cells, to be produced in sufficient quantities to answer important biological questions.