High Affinity Antigen Recognition of the Dual Specific Variants of Herceptin Is Entropy-Driven in Spite of Structural Plasticity

The antigen-binding site of Herceptin, an anti-human Epidermal Growth Factor Receptor 2 (HER2) antibody, was engineered to add a second specificity toward Vascular Endothelial Growth Factor (VEGF) to create a high affinity two-in-one antibody bH1. Crystal structures of bH1 in complex with either antigen showed that, in comparison to Herceptin, this antibody exhibited greater conformational variability, also called “structural plasticity”. Here, we analyzed the biophysical and thermodynamic properties of the dual specific variants of Herceptin to understand how a single antibody binds two unrelated protein antigens. We showed that while bH1 and the affinity-improved bH1-44, in particular, maintained many properties of Herceptin including binding affinity, kinetics and the use of residues for antigen recognition, they differed in the binding thermodynamics. The interactions of bH1 and its variants with both antigens were characterized by large favorable entropy changes whereas the Herceptin/HER2 interaction involved a large favorable enthalpy change. By dissecting the total entropy change and the energy barrier for dual interaction, we determined that the significant structural plasticity of the bH1 antibodies demanded by the dual specificity did not translate into the expected increase of entropic penalty relative to Herceptin. Clearly, dual antigen recognition of the Herceptin variants involves divergent antibody conformations of nearly equivalent energetic states. Hence, increasing the structural plasticity of an antigen-binding site without increasing the entropic cost may play a role for antibodies to evolve multi-specificity. Our report represents the first comprehensive biophysical analysis of a high affinity dual specific antibody binding two unrelated protein antigens, furthering our understanding of the thermodynamics that drive the vast antigen recognition capacity of the antibody repertoire

Translation Levels Control Multi-Spanning Membrane Protein Expression

Attempts to express eukaryotic multi-spanning membrane proteins at high-levels have been generally unsuccessful. In order to investigate the cause of this limitation and gain insight into the rate limiting processes involved, we have analyzed the effect of translation levels on the expression of several human membrane proteins in Escherichia coli (E. coli). These results demonstrate that excessive translation initiation rates of membrane proteins cause a block in protein synthesis and ultimately prevent the high-level accumulation of these proteins. Moderate translation rates allow coupling of peptide synthesis and membrane targeting, resulting in a significant increase in protein expression and accumulation over time. The current study evaluates four membrane proteins, CD20 (4-transmembrane (TM) helixes), the G-protein coupled receptors (GPCRs, 7-TMs) RA1c and EG-VEGFR1, and Patched 1 (12-TMs), and demonstrates the critical role of translation initiation rates in the targeting, insertion and folding of integral membrane proteins in the E. coli membrane

Bionic bodies, posthuman violence and the disembodied criminal subject

This article examines how the so-called disembodied criminal subject is given structure and form through the law of homicide and assault. By analysing how the body is materialised through the criminal law’s enactment of death and injury, this article suggests that the biological positioning of these harms of violence as uncontroversial, natural, and universal conditions of being ‘human’ cannot fully appreciate what makes violence wrongful for us, as embodied entities. Absent a theory of the body, and a consideration of corporeality, the criminal law risks marginalising, or altogether eliding, experiences of violence that do not align with its paradigmatic vision of what bodies can and must do when suffering its effects. Here I consider how the bionic body disrupts the criminal law’s understanding of human violence by being a body that is both organic and inorganic, and capable of experiencing and performing violence in unexpected ways. I propose that a criminal law that is more receptive to the changing, technologically mediated conditions of human existence would be one that takes the corporeal dimensions of violence more seriously and, as an extension of this, adopts an embodied, embedded, and relational understanding of human vulnerability to violence

Genetic diversity in local cultivars of garden pea (Pisum sativum L.) conserved on farm and in historical collections

During a national Swedish collection mission of vegetable varieties conserved on farm more than 70 pea accessions were obtained, many of which had been grown locally for more than 100 years. In spite of a likely origin in the multitude of obsolete commercial pea varieties available on the Swedish seed market in the nineteenth century, the rediscovered local cultivars have lost their original names and cultivar identity while being maintained on farm. To analyze genetic diversity in the repatriated material, 20 accessions were genotyped with twelve SSR markers and compared with 15 obsolete cultivars kept in genebanks and 13 cultivars preserved as non-viable seeds collected in 1877-1918. Most of the local cultivars were genetically distinct from each other, and in only a few cases could a possible origin in a tested obsolete cultivar be suggested. These results reflect the wide diversity of pea cultivars present in Sweden during the nineteenth century. Both between and within accession genetic diversity was larger among the historical samples of obsolete cultivars compared to local cultivars and cultivars preserved in genebanks, indicating genetic erosion over time both in genebanks and during conservation on farm. The constraints on identifying and verifying historical cultivars using genetic markers are discussed

Setting goals with patients at risk of malnutrition : A focus group study with clinical dietitians

Objective: Setting goals collaboratively with patients is a key aspect in shared decision-making (SDM) in malnutrition interventions. The aim, therefore, was to gain an understanding of clinical dietitians' reflections regarding the process of goal-setting with patients at risk of malnutrition. Methods: Six semi-structured audio-recorded focus group discussions were held with registered dietitians (n = 29) from primary healthcare and hospitals in Sweden. Focus group transcripts were analysed thematically to find patterns in the data and identify themes. Results: Dietitians expressed striving to explore patients' narratives, capabilities, and resources before deciding on goals. They described different strategies in counseling patients and a lack of patient participation in the goal setting. They emphasized the difficulties of setting feasible goals due to discrepancies between their clinically oriented goals and patients' personal goals. Conclusion: Findings highlight a gap in the process of setting goals for patients at risk of malnutrition, where patients' participation was lacking. Education in SDM, and strategies and tools to support dietitians in involving patients in goal-setting, are required to bridge the gap and promote person-centeredness. Practice implications: Findings may be further used to develop tools and strategies, and design studies on the implementation of and education in SDM and goal-setting for malnutrition interventions

Expression and folding of CD20 in the <i>E. coli</i> inner membrane.

<p>Cell surface expression and orientation of CD20 was assessed from spheroplasts of <i>E. coli</i> cells expressing either Uni-CD20 (blue), LE-CD20 (green) or an empty control vector (red) treated with Alexa-488 conjugated anti-CD20 antibody to the extracellular loop of CD20 and analyzed by flow cytometry.</p

Ligand binding to the GPCR, LE-EG-VEGFR1.

<p><i>E. coli</i> membrane proteoliposomes were treated with thrombin to remove the LE-leader and incubated overnight at 4°C with EG-VEGF in PBS. Pelleted membranes were separated by SDS-PAGE and developed by immuno-blot using an anti-EG VEGF antibody. Samples are: lane 1) pBR322 negative control; 2) LE-EG-VEGFR1, N-terminal FLAG; 3) LE-EG-VEGFR1, C-terminal FLAG. The location of EG-VEGF (molecular weight 9 kDa) is indicated by an arrow.</p

Improved cell growth and general accumulation of integral membrane proteins using a dually regulated promoter.

<p>(<b>A</b>) Restricted <i>E. coli</i> growth in LB with the <i>phoA</i>-RA1c construct is relieved by using the <i>tphac</i> promoter, which reduces basal level expression. A 24-hour growth curve shows the empty pBR322 vector control (blue triangles), <i>phoA</i>-RA1c expression construct (green diamonds), <i>tphac</i>-RA1c expression construct (red circles) and <i>phoA</i>-EGFL7 as a non-membrane protein control (brown squares). (<b>B</b>) A representative western blot of RA1c expression from the <i>phoA</i> promoter is shown following induction by phosphate depletion when the cells reach approximately 2 OD₆₀₀ (time 0). Maximum expression is reached within two hours post induction. By 6 hours, aggregation has begun and by twelve hours almost all the protein has moved from the monomer band to high molecular weight aggregate. Basal expression is shown after overnight growth in LB medium (LBON). The western blot was probed with an HRP coupled anti-his antibody. (<b>C</b>) A comparison of basal expression in LB of the GPCR proteins, RA1c and EG-VEGFR1, from the <i>phoA</i> and <i>tphac</i> promoters by western blot analysis. The <i>phoA</i> constructs show significant accumulation levels of the membrane proteins while the <i>tphac</i> constructs have reduced the accumulation to background levels. The arrow points to the monomer protein band.</p