Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization¹ . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used² . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions³ . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies^1,2.Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding⁴ , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A⁵, a small, bispecific molecule with affinity for both HSA and the novel target was identified⁶ .The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination^1,7.     

Evidence for a factor in pig adipose tissue controlling the specificity of the acyltransferase(s) of triacylglycerol synthesis.

The proportion of palmitate at position 2 of triacylglycerols synthesized by microsome-cytosol preparations of rat, mouse, or chicken adipose tissue was found to be markedly increased by the addition of an inactivated preparation of pig adipose tissue microsomes. A phosphate extract of pig adipose tissue microsomes was also active in directing palmitate to position 2 of rat triacylglycerols. The proportion of palmitate at position 2 of triacylglycerols synthesized was reduced by prior repeated extraction of the pig adipose tissue microsomes with phosphate. The pretreatment specificity was restored when the extract was added back to the microsomes. These observations support the proposal that the unusually high proportion of palmitate at position 2 of lard results from the action of a factor controlling the specificity of the acyltransferase(s) from pig adipose tissue.     

Simulating viability of a spadefoot toad Pelobates fuscus metapopulation in a landscape fragmented by a road

A stochastic simulation model allowing for demographic and environmental stochasticity was developed in order to predict the dynamics of a Pelobates fuscus metapopulation. The metapopulation (consisting of ca 1000 adult individuals) was intensively studied in the field for a period of four years and the simulation model was parameterised (sex ratio, age‐specific survival rates, fecundity and dispersal between subpopulations) using field data. A sensitivity analysis revealed that a change in the juvenile yearly survival rate has a relatively larger effect on subpopulation persistence than adult survival rate and fecundity rate have. The probability of subpopulation persistence for one hundred years increased from 0 to 0.6 with a change in juvenile yearly survival rate from 0.35 to 0.40. Varying dispersal rate (from 0 to 1% of the individuals in a subpopulation moving to another subpopulation within a year) showed that four of the five subpopulations are dependent on the last one for persistence, indicating a source‐sink structure of the metapopulation. The subpopulation with the highest estimated juvenile survival has a far higher persistence probability than the others; they in turn would go extinct were it not for the occasional input of individuals from the source subpopulation. The source‐sink structure was also apparent when simulating the isolation effect of the road: persistence of the subpopulation isolated by the road decreases markedly with only a 20% decrease in the number of individuals dispersing to this pond. Environmental stochasticity decreases persistence time of the source subpopulation, but increases persistence time of the unstable ones. This is probably due to the presence of overcompensating density‐dependent factors affecting the subpopulations and to the more general effect of stochasticity: it may temporarily change reproduction and mortality rates, increase time to extinction for unstable subpopulations through a rescue effect; and decrease time to extinction for the more stable subpopulations.     

Cell-based optimization of novel benzamides as potential antimalarial leads

Screening our in-house compound collection using a cell based Plasmodium falciparum proliferation assay we discovered a known pan-kinase inhibitor scaffold as a hit. Further optimization of this series led us to a novel benzamide scaffold which was devoid of human kinase activity while retaining its antiplasmodial activity. The evolution of this compound series leading to optimized candidates with good cellular potency against multiple strains as well as decent in vivo profile is described in this Letter.     

The Effect of Alternative Summary Statistics for Communicating Risk Reduction on Decisions about Taking Statins: A Randomized Trial

Background

While different ways of presenting treatment effects can affect health care decisions, little is known about which presentations best help people make decisions consistent with their own values. We compared six summary statistics for communicating coronary heart disease (CHD) risk reduction with statins: relative risk reduction and five absolute summary measures—absolute risk reduction, number needed to treat, event rates, tablets needed to take, and natural frequencies.

Methods and Findings

We conducted a randomized trial to determine which presentation resulted in choices most consistent with participants'' values. We recruited adult volunteers who participated through an interactive Web site. Participants rated the relative importance of outcomes using visual analogue scales (VAS). We then randomized participants to one of the six summary statistics and asked them to choose whether to take statins based on this information. We calculated a relative importance score (RIS) by subtracting the VAS scores for the downsides of taking statins from the VAS score for CHD. We used logistic regression to determine the association between participants'' RIS and their choice. 2,978 participants completed the study. Relative risk reduction resulted in a 21% higher probability of choosing to take statins over all values of RIS compared to the absolute summary statistics. This corresponds to a number needed to treat (NNT) of 5; i.e., for every five participants shown the relative risk reduction one additional participant chose to take statins, compared to the other summary statistics. There were no significant differences among the absolute summary statistics in the association between RIS and participants'' decisions whether to take statins. Natural frequencies were best understood (86% reported they understood them well or very well), and participants were most satisfied with this information.

Conclusions

Presenting the benefits of taking statins as a relative risk reduction increases the likelihood of people accepting treatment compared to presenting absolute summary statistics, independent of the relative importance they attach to the consequences. Natural frequencies may be the most suitable summary statistic for presenting treatment effects, based on self-reported preference, understanding of and satisfaction with the information, and confidence in the decision.

Clinical Trials Registration

ISRCTN85194921 Please see later in the article for the Editors'' Summary     

Differences in habitat quality explain nestedness in a land snail meta-community

We set up two alternative hypotheses on how environmental variables could foster nestedness; one of “nested habitats” and another of “nested habitat quality”. The former hypothesis refers to situations where the nestedness of species depends on a nestedness of discrete habitats. The latter considers situations where all species in an assemblage increase in abundance along the same environmental gradient, but differ in specialisation or tolerance. We tested whether litter‐dwelling land snails (terrestrial gastropods) in boreal riparian forest exhibited a nested community structure, whether such a pattern was related to differences in environmental variables among sites, and which of the two hypotheses that best could account for the found pattern. We sampled litter from 100 m² plots in 29 mature riparian forest sites along small streams in the boreal zone of Sweden. The number of snail species varied between 3 and 14 per site. Ranking the species‐by‐site matrix by PCA scores of the first ordination axis revealed a similarly significant nested pattern as when the matrix was sorted by number of species, showing that the species composition in this meta‐community can be properly described as nested. Several environmental variables, most notably pH index, were correlated with the first PCA axis. All but two species had positive eigenvectors in the PCA ordination and the abundance increased considerably along the gradient for most of the species implying that the hypothesis of “nested habitats” was rejected in favour of the “nested habitat quality” hypothesis. Analyses of nestedness have seldom been performed on equal sized plots, and our study shows the importance of understanding that variation in environmental variables among sites can result in nested communities. The conservation implications are different depending on which of our two hypotheses is supported; a conservation focus on species “hotspots” is more appropriate if the communities are nested because of “nested habitat quality”.     

Natural variation in the microcystin synthetase operon mcyABC and impact on microcystin production in Microcystis strains

Toxic Microcystis strains often produce several isoforms of the cyclic hepatotoxin microcystin, and more than 65 isoforms are known. This has been attributed to relaxed substrate specificity of the adenylation domain. Our results show that in addition to this, variability is also caused by genetic variation in the microcystin synthetase genes. Genetic characterization of a region of the adenylation domain in module mcyB1 resulted in identification of two groups of genetic variants in closely related Microcystis strains. Sequence analyses suggested that the genetic variation is due to recombination events between mcyB1 and the corresponding domains in mcyC. Each variant could be correlated to a particular microcystin isoform profile, as identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Among the Microcystis species studied, we found 11 strains containing different variants of the mcyABC gene cluster and 7 strains lacking the genes. Furthermore, there is no concordance between the phylogenies generated with mcyB1, 16S ribosomal DNA, and DNA fingerprinting. Collectively, these results suggest that recombination between imperfect repeats, gene loss, and horizontal gene transfer can explain the distribution and variation within the mcyABC operon.     

Structure and kinetic properties of Paracoccus pantotrophus cytochrome cd1 nitrite reductase with the d1 heme active site ligand tyrosine 25 replaced by serine

The 1.4-A crystal structure of the oxidized state of a Y25S variant of cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus is described. It shows that loss of Tyr(25), a ligand via its hydroxy group to the iron of the d(1) heme in the oxidized (as prepared) wild-type enzyme, does not result in a switch at the c heme of the unusual bishistidinyl coordination to the histidine/methionine coordination seen in other conformations of the enzyme. The Ser(25) side chain is seen in two positions in the d(1) heme pocket with relative occupancies of approximately 7:3, but in neither case is the hydroxy group bound to the iron atom; instead, a sulfate ion from the crystallization solution is bound between the Ser(25) side chain and the heme iron. Unlike the wild-type enzyme, the Y25S mutant is active as a reductase toward nitrite, oxygen, and hydroxylamine without a reductive activation step. It is concluded that Tyr(25) is not essential for catalysis of reduction of any substrate, but that the requirement for activation by reduction of the wild-type enzyme is related to a requirement to drive the dissociation of this residue from the active site. The Y25S protein retains the d(1) heme less well than the wild-type protein, suggesting that the tyrosine residue has a role in stabilizing the binding of this cofactor.