Control Features (control + feature)

Distribution by Scientific Domains

Selected Abstracts

Exploration of twin-arginine translocation for expression and purification of correctly folded proteins in Escherichia coli

Adam C. Fisher
Summary Historically, the general secretory (Sec) pathway of Gram-negative bacteria has served as the primary route by which heterologous proteins are delivered to the periplasm in numerous expression and engineering applications. Here we have systematically examined the twin-arginine translocation (Tat) pathway as an alternative, and possibly advantageous, secretion pathway for heterologous proteins. Overall, we found that: (i) export efficiency and periplasmic yield of a model substrate were affected by the composition of the Tat signal peptide, (ii) Tat substrates were correctly processed at their N-termini upon reaching the periplasm and (iii) proteins fused to maltose-binding protein (MBP) were reliably exported by the Tat system, but only when correctly folded; aberrantly folded MBP fusions were excluded by the Tat pathway's folding quality control feature. We also observed that Tat export yield was comparable to Sec for relatively small, well-folded proteins, higher relative to Sec for proteins that required cytoplasmic folding, and lower relative to Sec for larger, soluble fusion proteins. Interestingly, the specific activity of material purified from the periplasm was higher for certain Tat substrates relative to their Sec counterparts, suggesting that Tat expression can give rise to relatively pure and highly active proteins in one step. [source]

Mining mammalian genomes for folding competent proteins using Tat-dependent genetic selection in Escherichia coli

PROTEIN SCIENCE, Issue 12 2009
Hyung-Kwon Lim
Abstract Recombinant expression of eukaryotic proteins in Escherichia coli is often limited by poor folding and solubility. To address this problem, we employed a recently developed genetic selection for protein folding and solubility based on the bacterial twin-arginine translocation (Tat) pathway to rapidly identify properly folded recombinant proteins or soluble protein domains of mammalian origin. The coding sequences for 29 different mammalian polypeptides were cloned as sandwich fusions between an N-terminal Tat export signal and a C-terminal selectable marker, namely ,-lactamase. Hence, expression of the selectable marker and survival on selective media was linked to Tat export of the target mammalian protein. Since the folding quality control feature of the Tat pathway prevents export of misfolded proteins, only correctly folded fusion proteins reached the periplasm and conferred cell survival. In general, the ability to confer growth was found to relate closely to the solubility profile and molecular weight of the protein, although other features such as number of contiguous hydrophobic amino acids and cysteine content may also be important. These results highlight the capacity of Tat selection to reveal the folding potential of mammalian proteins and protein domains without the need for structural or functional information about the target protein. [source]

Biologically inspired climbing with a hexapedal robot

M. J. Spenko
This paper presents an integrated, systems-level view of several novel design and control features associated with the biologically inspired, hexapedal, RiSE (Robots in Scansorial Environments) robot. RiSE is the first legged machine capable of locomotion on both the ground and a variety of vertical building surfaces including brick, stucco, and crushed stone at speeds up to 4 cm/s, quietly and without the use of suction, magnets, or adhesives. It achieves these capabilities through a combination of bioinspired and traditional design methods. This paper describes the design process and specifically addresses body morphology, hierarchical compliance in the legs and feet, and sensing and control systems that enable robust and reliable climbing on difficult surfaces. Experimental results illustrate the effects of various behaviors on climbing performance and demonstrate the robot's ability to climb reliably for long distances. 2008 Wiley Periodicals, Inc. [source]

Mouse Strain and Injection Site are Crucial for Detecting Linked Suppression in Transplant Recipients by Trans-Vivo DTH Assay

W.J. Burlingham
Chemokine-driven accumulation of lymphocytes, mononuclear and polymorphonuclear proinflammatory cells in antigenic tissue sites is a key feature of several types of T-cell-dependent autoimmunity and transplant rejection pathology. It is now clear that the immune system expends considerable energy to control this process, exemplified by the sequential layers of regulatory cell input, both innate and adaptive, designed to prevent a classical Type IV or ,delayed-type' hypersensitivity (DTH) reaction from occurring in the visual field of the eye. Yet, despite an abundance of in vitro assays currently available to the human T-cell immunologist, none of them adequately models the human DTH response and its various control features. The theme of this article is that it is relatively easy to model the effector side of the human DTH response with xenogeneic adoptive transfer models. However, we show that in order to detect inhibition of a recall DTH in response to colocalized donor antigen (linked suppression),a characteristic feature of peripheral tolerance to an organ transplant,both the challenge site and the immunocompetence of the mouse adoptive host are critical factors limiting the sensitivity of the trans-vivo DTH test. [source]

Cognitive function and symptoms in adults and adolescents in relation to rf radiation from UMTS base stations

Ingunn S. Riddervold
Abstract There is widespread public concern about the potential adverse health effects of mobile phones in general and their associated base stations in particular. This study was designed to investigate the acute effects of radio frequency (RF) electromagnetic fields (EMF) emitted by the Universal Mobile Telecommunication System (UMTS) mobile phone base stations on human cognitive function and symptoms. Forty adolescents (15,16 years) and 40 adults (25,40 years) were exposed to four conditions: (1) sham, (2) a Continuous Wave (CW) at 2140 MHz, (3) a signal at 2140 MHz modulated as UMTS and (4) UMTS at 2140 MHz including all control features in a randomized, double blinded cross-over design. Each exposure lasted 45 min. During exposure the participants performed different cognitive tasks with the Trail Making B (TMB) test as the main outcome and completed a questionnaire measuring self reported subjective symptoms. No statistically significant differences between the UMTS and sham conditions were found for performance on TMB. For the adults, the estimated difference between UMTS and sham was ,3.2% (,9.2%; 2.9%) and for the adolescents 5.5% (,1.1%; 12.2%). No significant changes were found in any of the cognitive tasks. An increase in ,headache rating' was observed when data from the adolescents and adults were combined (P,=,0.027), an effect that may be due to differences at baseline. In conclusion, the primary hypothesis that UMTS radiation reduces general performance in the TMB test was not confirmed. However, we suggest that the hypothesis of subjective symptoms and EMF exposure needs further research. Bioelectromagnetics 29:257,267, 2008. 2007 Wiley-Liss, Inc. [source]