A general approach to antibody thermostabilization

Antibody engineering to enhance thermostability may enable further application and ease of use of antibodies across a number of different areas. A modified human IgG framework has been developed through a combination of engineering approaches, which can be used to stabilize antibodies of diverse specificity. This is achieved through a combination of complementarity-determining region (CDR)-grafting onto the stable framework, mammalian cell display and in vitro somatic hypermutation (SHM). This approach allows both stabilization and maturation to affinities beyond those of the original antibody, as shown by the stabilization of an anti-HA33 antibody by approximately 10°C and affinity maturation of approximately 300-fold over the original antibody. Specificities of 10 antibodies of diverse origin were successfully transferred to the stable framework through CDR-grafting, with 8 of these successfully stabilized, including the therapeutic antibodies adalimumab, stabilized by 9.9°C, denosumab, stabilized by 7°C, cetuximab stabilized by 6.9°C and to a lesser extent trastuzumab stabilized by 0.8°C. This data suggests that this approach may be broadly useful for improving the biophysical characteristics of antibodies across a number of applications.     

Simultaneous Extraction of RNA and DNA from Rat Liver with Cupric Sulfate

Homogenization of fresh rat livers in 0.5mM cupric sulfate and 0.5% sodium dodecyl sulfate yielded both RNA and DNA in the aqueous phase after treatment vith phenol at 0–4°C. Effective deproteinization was achieved by three additional phenol treatments. Nucleic acids vere freed from Cu²⁺by repeated precipitation with ethanol in the presence of EDTA. The final yield was 6–7 mg/g of liver, of which about 20% was DNA and 11% tRNA. Physicochenical studies showed that pure tRNA, undegraded rRKA (30S, 18S) and native DNA (s°_{20, w} = 24.4S) were isolated by this procedure.     

Importance of non-conserved distal carboxyl terminal amino acids in two peptidases belonging to the M1 family: Thermoplasma acidophilum Tricorn interacting factor F2 and Escherichia coli Peptidase N

Enzymes belonging to the M1 family play important cellular roles and the key amino acids (aa) in the catalytic domain are conserved. However, C-terminal domain aa are highly variable and demonstrate distinct differences in organization. To address a functional role for the C-terminal domain, progressive deletions were generated in Tricorn interacting factor F2 from Thermoplasma acidophilum (F2) and Peptidase N from Escherichia coli (PepN). Catalytic activity was partially reduced in PepN lacking 4 C-terminal residues (PepNΔC4) whereas it was greatly reduced in F2 lacking 10 C-terminal residues (F2ΔC10) or PepN lacking eleven C-terminal residues (PepNΔC11). Notably, expression of PepNΔC4, but not PepNΔC11, in E. coliΔpepN increased its ability to resist nutritional and high temperature stress, demonstrating physiological significance. Purified C-terminal deleted proteins demonstrated greater sensitivity to trypsin and bound stronger to 8-amino 1-napthalene sulphonic acid (ANS), revealing greater numbers of surface exposed hydrophobic aa. Also, F2 or PepN containing large aa deletions in the C-termini, but not smaller deletions, were present in high amounts in the insoluble fraction of cell extracts probably due to reduced protein solubility. Modeling studies, using the crystal structure of E. coli PepN, demonstrated increase in hydrophobic surface area and change in accessibility of several aa from buried to exposed upon deletion of C-terminal aa. Together, these studies revealed that non-conserved distal C-terminal aa repress the surface exposure of apolar aa, enhance protein solubility, and catalytic activity in two soluble and distinct members of the M1 family.     

High throughput detection of antibody self-interaction by bio-layer interferometry

Self-interaction of an antibody may lead to aggregation, low solubility or high viscosity. Rapid identification of highly developable leads remains challenging, even though progress has been made with the introduction of techniques such as self-interaction chromatography (SIC) and cross-interaction chromatography (CIC). Here, we report a high throughput method to detect antibody clone self-interaction (CSI) using bio-layer interferometry (BLI) technology. Antibodies with strong self-interaction responses in the CSI-BLI assay also show delayed retention times in SIC and CIC. This method allows hundreds of candidates to be screened in a matter of hours with minimal material consumption.     

Oxygen solubility in evaporated seawater as a function of temperature and salinity

Most studies of oxygen solubility values for high salinity conditions have used synthetic solutions. The object of this study is therefore to propose an equation, valid for high salinity conditions, based on the analysis of oxygen saturation in evaporated seawater. In this study, the solubility of oxygen in evaporated seawater has been determined over a temperature range of 8–35°C and with salinity values of up to 133‰. Based on experimental data, an equation is proposed that introduces a S ² (salinity) term, at 1 atm pressure, giving increased importance to salinity. The equation provides a valid means of predicting the amount of dissolved oxygen in this range of temperatures and salinities. In addition, for high salinity conditions, with this equation there is no need to extrapolate other established equations, which are less accurate at salinities higher than 40‰. The use of the proposed equation offers a more precise way of calculating oxygen solubility in seawater at high salinity values (up to 133‰), and small deviations from experimental values, of the order of 2 μmol kg⁻¹, are obtained. Handling editor: J. Melack     

Analysis of protein aggregation kinetics using short amino acid peptide tags

Understanding protein solubility, and consequently aggregation, is an important issue both from an academic and a biotechnological application viewpoints. Here we report the effects of 10 representative amino acids on the aggregation kinetics of proteins. The effects were determined by measuring the solubility of a simplified bovine pancreatic trypsin inhibitor (BPTI) variant, to which short artificial tags containing the amino acid of interest were added at its C-terminus. We determined the solubility of the tagged variants as a function of equilibration time (20 min to 48 h) and total protein concentration ranging from 0.10 mg/ml to 25.0 mg/ml. We observed, as anticipated, that proteins precipitated when the total protein concentration exceeded a critical value. However, when the total protein concentration was further increased, the apparent solubility reached a concentration above the critical value, and slowly decreased to a value under the critical concentration upon increasing the equilibration period. We rationalized these observations by identifying three different solubility values, the “transient solubility (TS)”, the “aggregation initiation concentration (AIC)” and the “long-term solubility (LS)”. AIC and LS are parameters determined essentially by the amino acid types composing the tags and could be considered as an amino acid's intrinsic property. On the other hand, TS is an apparent solubility that is measured after some (20 min in our case) equilibration time and is often considered as the “solubility” of the protein. Similar aggregation kinetic patterns were observed with natural proteins, indicating the generality of the observations made using our model protein.     

Solubility evaluation of murine hybridoma antibodies

The successful development of antibody therapeutics depends on the molecules having properties that are suitable for manufacturing, as well as use by patients. Because high solubility is a desirable property for antibodies, screening for solubility has become an essential step during the early candidate selection process. In considering the screening process, we formed a hypothesis that hybridoma antibodies are filtered by nature to possess high solubility and tested this hypothesis using a large number of murine hybridoma-derived antibodies. Using the cross-interaction chromatography (CIC) method, we screened the solubility of 92 murine hybridoma-derived monoclonal antibodies and found that all of these molecules exhibited CIC profiles that are indicative of high solubility (>100mg/mL). Further investigations revealed that variable region N-linked glycosylation or isoelectric parameters are unlikely to contribute to the high solubility of these antibodies. These results support the general hypothesis that hybridoma monoclonal antibodies are highly soluble.     

Distribution of heavy metals in surface sediments of the Bacochibampo Bay,Sonora, Mexico

Abstract

The aim of this study was to determine the levels of heavy metals (Al, Cd, Cr, Cu, Fe, Mn and Pb) in the geochemical fractions of the coastal surface sediments from the Bacochibampo Bay in Sonora, Mexico. Two surveys were conducted (March and September) during 2004, at eight sampling stations inside the bay, and in three natural effluents discharged into this bay. The extraction of metals was carried out using a microwave oven method and the quantification was done by atomic absorption spectro-photometry. The highest detected concentration of total heavy metals in sediments was: Fe>Al>Mn>Pb>Cr>Cu>Cd, with the following concentration values: Fe (1.72%), Al (1.03%), Mn (416.31 mg kg^?1), Pb (11.73 mg kg^?1), Cr (11.41 mg kg^–1), Cu (6.78 mg kg^–1) and Cd (1.33 mg kg^–1). The levels of total heavy metals (Al, Cr, Cu, Fe, Mn and Pb) were much less than the lowest observable effect level (LEL) which indicates that the sediments were not from polluted areas and that the origin of the metals was due to natural conditions. However, concentrations of Cd were much higher than the low effect level (LEL), over 40% of metal was detected in the exchangeable fraction and carbonates. The normalisation study showed a high degree of enrichment of Cd in all the sampling stations in the Bacochibampo Bay (samples EF 34–87) and in the natural flows that discharge into this bay (samples EF 22–35%), which exceeds by several orders of magnitude the value of sample EF 1, which indicates that Cd is anthropogenically induced. Based on these results, it is important that precautionary measures are established, since the deposited Cd in these fractions may be potentially toxic, due to the physicochemical changes that occur in the environment. Thus, future studies will focus on identifying problems involved with Cd bioaccumulation in different trophic levels.     

The availability of soil boron fractions to olive trees and barley and their relationships to soil properties

Soil boron occurring in various forms was correlated with boron contents in the leaves of olive trees in 51 and in barley leaves in 20 soils. The amounts of boron: in soil solution (CwsB), non specifically adsorbed (NsaB), specifically adsorbed (SaB), occluded in Mn oxides (MnoB), occluded in amorphous Fe-Al oxyhydroxides (FeoB), were correlated with soil properties such as: organic matter content, pH, free aluminum and iron oxyhydroxides (Ald, Fed), amorphous aluminum and iron oxyhydroxides (Alo, Feo). The later correlation studies were conducted on a total number of 153 soil samples inclusive of the soils used for the plant uptake investigations.The results show that the boron contents in the leaves of olive trees were significantly correlated with FeoB, CwsB, SaB and MnoB but with CwsB, NsaB, SaB and FeoB in barley leaves. The respective correlation coefficients suggest that available forms of soil boron vary with plant species.The hot water soluble boron was significantly correlated with all the fractions of boron studied except MnoB, confirming its value as a measure of available soil boron. However, the non significant (a=0.05) correlation with MnoB, which show significant correlation with the contents in the leaves of olive trees and barley, suggest that this procedure does not extract significant amounts of available B held in Mn oxyhydroxides.The highest amount of CwsB originated from the Cws B and to a lesser degree from the SaB and NsaB. The correlation among the boron held in the forms studied and the selected soil properties were either no significant or significant with the highest correlation found between FeoB and pH and Feo.