Heterologous expression of naturally evolved putative de novo proteins with chaperones

Over the past decade, evidence has accumulated that new protein‐coding genes can emerge de novo from previously non‐coding DNA. Most studies have focused on large scale computational predictions of de novo protein‐coding genes across a wide range of organisms. In contrast, experimental data concerning the folding and function of de novo proteins are scarce. This might be due to difficulties in handling de novo proteins in vitro, as most are short and predicted to be disordered. Here, we propose a guideline for the effective expression of eukaryotic de novo proteins in Escherichia coli. We used 11 sequences from Drosophila melanogaster and 10 from Homo sapiens, that are predicted de novo proteins from former studies, for heterologous expression. The candidate de novo proteins have varying secondary structure and disorder content. Using multiple combinations of purification tags, E. coli expression strains, and chaperone systems, we were able to increase the number of solubly expressed putative de novo proteins from 30% to 62%. Our findings indicate that the best combination for expressing putative de novo proteins in E. coli is a GST‐tag with T7 Express cells and co‐expressed chaperones. We found that, overall, proteins with higher predicted disorder were easier to express.StatementToday, we know that proteins do not only evolve by duplication and divergence of existing proteins but also arise from previously non‐coding DNA. These proteins are called de novo proteins. Their properties are still poorly understood and their experimental analysis faces major obstacles. Here, we aim to present a starting point for soluble expression of de novo proteins with the help of chaperones and thereby enable further characterization.     

Control of the Cranio-Cervical System During Feeding in Birds

The avian neck is a complex, kinematically redundant system,which plays a role during inter alia food prehension and manipulation.Kinematical analysis shows that chickens (Gallus domesticus)move their vertebrae according to a geometric principle thatmaximizes angular rotation efficiency. The movement patternshows simultaneous rotations in some joints, while not in theothers. Anseriformes show a pattern of successive, rather thansimultaneous rotations in the rostral part of the neck. A kinematicalmodel indicates that the geometric principle produces an anseriform-likepattern only if a constraint on the movement of the caudal vertebraeis introduced. The strength of this constraint, required fora realistic simulation, is related to the amount of stretchin the long dorsal neck muscles (M. biventer and M. longus collidorsalis), which have a different configuration in Anseriformescompared to the chicken. To investigate whether the differencein movement pattern is a result of differences in anatomy only,or also of differences in neuromotor patterns, the EMG-patternsof the neck muscles of the mallard and chicken during drinkingand pecking were studied. Considerable overlap in the activityof antagonists is found in mallards, but not in chickens. Musclesin the rostral part of the neck are activated successively inmallards, but simultaneously in chickens. We conclude that thedifference in movement patterning between chickens and Anseriformes,results from both a difference in the control system of theneck, and a difference in the anatomy. The anseriform patternis found in water as well as on land, which suggests that neckmovement in both environments is controlled by the same neuromotorpatterns. The modifications in motor control system and anatomyof the Anseriformes may have evolved as an adaptation to aquaticfeeding, since the anseriform pattern is energetically morebeneficial in an aquatic environment than on land.     

Further observations on the specificity of antigen 5 of Echinococcus granulosus.

The presence of IgE antibodies to antigen 5 of Echinococcus granulosus was detected by means of radioimmunoelectrophoresis in the sera of two of six patients infected with E. multilocularis. Sera from three of these patients gave a precipitin band in gel diffusion tests identical to that produced by a monospecific rabbit anti-E. granulosus antigen 5 serum, when tested against whole hydatid fluid. Sera from 19 individuals infected with Fasciola hepatica, 20 with Schistosoma mansoni, and 5 with with Taenia saginata showed no detectable antibodies against antigen 5 of E. granulosus, The monospecific rabbit anti-E. granulosus antigen 5 serum did not react in immunodiffusion with homologous antigen when absorbed with either 4 mg/ml of whole hydatid fluid or with 200 mg/ml of a soluble E. multilocularis extract. Absorption of the monospecific antiserum with crude antigens of either F. hepatica, Onchocerca volvulus, S. mansoni, or T. saginata did not abolish the reaction with antigen 5. It appears, therefore, that antigen 5 can no longer be considered specific for E. granulosus, but is also present in E. multilocularis. In the light of this observation, some reevaluation of immunodiagnostic tests in hydatid disease will be necessary.     

Zonal distribution of peroxisomal 3-oxoacyl-CoA thiolase mRNA in liver from rats treated with di-(2-ethylhexyl)phthalate

Treatment of rats with di-(2-ethylhexyl)phthalate leads to a dramatic increase in peroxisomal 3-oxoacyl-CoA thiolase RNA, the concentration being higher in the pericentral than in periportal hepatocytes. These findings indicate that the production of peroxisomal thiolase and the zonal distribution of the enzyme are regulated at a pretranslational level.     

Identification and purification of the specific antigen of Paracoccidioides brasiliensis responsible for immunoelectrophoretic band E.

A new purified antigen (E2) of Paracoccidioides brasiliensis mycelial growth phase was isolated by immunoadsorption from a crude metabolic soluble extract of the fungus. The antiserum prepared in a rabbit by inoculation of E2 antigen developed only one immunodiffusion line with the crude metabolic extract. Findings on immunological analysis showed that E2 antigen is the antigenic component of immunoelectrophoretic band E. The isolated antigens did not possess detectable alkaline phosphatase activity. It reacted in immunodiffusion tests with all the sera (14/14) from P. brasiliensis infected patients containing precipitating antibodies.     

Notiz über Azotobacter agilis Beijerinck

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