Streamlined protein expression and purification using cleavable self-aggregating tags

<p>Abstract</p> <p>Background</p> <p>Recombinant protein expression and purification remains a fundamental issue for biotechnology. Recently we found that two short self-assembling amphipathic peptides 18A (EWLKAFYEKVLEKLKELF) and ELK16 (LELELKLKLELELKLK) can induce the formation of active protein aggregates in <it>Escherichia coli </it>(<it>E. coli</it>), in which the target proteins retain high enzymatic activities. Here we further explore this finding to develop a novel, facile, matrix-free protein expression and purification approach.</p> <p>Results</p> <p>In this paper, we describe a streamlined protein expression and purification approach by using cleavable self-aggregating tags comprising of one amphipathic peptide (18A or ELK16) and an intein molecule. In such a scheme, a target protein is first expressed as active protein aggregate, separated by simple centrifugation, and then released into solution by intein-mediated cleavage. Three target proteins including lipase A, amadoriase II and β-xylosidase were used to demonstrate the feasibility of this approach. All the target proteins released after cleavage were highly active and pure (over 90% in the case of intein-ELK16 fusions). The yields were in the range of 1.6-10.4 μg/mg wet cell pellet at small laboratory scale, which is comparable with the typical yields from the classical his-tag purification, the IMPACT-CN system (New England Biolabs, Beverly, MA), and the ELP tag purification scheme.</p> <p>Conclusions</p> <p>This tested single step purification is capable of producing proteins with high quantity and purity. It can greatly reduce the cost and time, and thus provides application potentials for both industrial scale up and laboratorial usage.</p

Active protein aggregates induced by terminally attached self-assembling peptide ELK16 in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>In recent years, it has been gradually realized that bacterial inclusion bodies (IBs) could be biologically active. In particular, several proteins including green fluorescent protein, β-galactosidase, β-lactamase, alkaline phosphatase, <smcaps>D</smcaps>-amino acid oxidase, polyphosphate kinase 3, maltodextrin phosphorylase, and sialic acid aldolase have been successfully produced as active IBs when fused to an appropriate partner such as the foot-and-mouth disease virus capsid protein VP1, or the human β-amyloid peptide Aβ42(F19D). As active IBs may have many attractive advantages in enzyme production and industrial applications, it is of considerable interest to explore them further.</p> <p>Results</p> <p>In this paper, we report that an ionic self-assembling peptide ELK16 (LELELKLK)₂was able to effectively induce the formation of cytoplasmic inclusion bodies in <it>Escherichia coli </it>(<it>E. coli</it>) when attached to the carboxyl termini of four model proteins including lipase A, amadoriase II, β-xylosidase, and green fluorescent protein. These aggregates had a general appearance similar to the usually reported cytoplasmic inclusion bodies (IBs) under transmission electron microscopy or fluorescence confocal microscopy. Except for lipase A-ELK16 fusion, the three other fusion protein aggregates retained comparable specific activities with the native counterparts. Conformational analyses by Fourier transform infrared spectroscopy revealed the existence of newly formed antiparallel beta-sheet structures in these ELK16 peptide-induced inclusion bodies, which is consistent with the reported assembly of the ELK16 peptide.</p> <p>Conclusions</p> <p>This has been the first report where a terminally attached self-assembling β peptide ELK16 can promote the formation of active inclusion bodies or active protein aggregates in <it>E. coli</it>. It has the potential to render <it>E. coli </it>and other recombinant hosts more efficient as microbial cell factories for protein production. Our observation might also provide hints for protein aggregation-related diseases.</p

Text Style Transfer Back-Translation

Back Translation (BT) is widely used in the field of machine translation, as it has been proved effective for enhancing translation quality. However, BT mainly improves the translation of inputs that share a similar style (to be more specific, translation-like inputs), since the source side of BT data is machine-translated. For natural inputs, BT brings only slight improvements and sometimes even adverse effects. To address this issue, we propose Text Style Transfer Back Translation (TST BT), which uses a style transfer model to modify the source side of BT data. By making the style of source-side text more natural, we aim to improve the translation of natural inputs. Our experiments on various language pairs, including both high-resource and low-resource ones, demonstrate that TST BT significantly improves translation performance against popular BT benchmarks. In addition, TST BT is proved to be effective in domain adaptation so this strategy can be regarded as a general data augmentation method. Our training code and text style transfer model are open-sourced.Comment: acl2023, 14 pages, 4 figures, 19 table

Small surfactant-like peptides can drive soluble proteins into active aggregates

<p>Abstract</p> <p>Background</p> <p>Inactive protein inclusion bodies occur commonly in <it>Escherichia coli </it>(<it>E. coli</it>) cells expressing heterologous proteins. Previously several independent groups have found that active protein aggregates or pseudo inclusion bodies can be induced by a fusion partner such as a cellulose binding domain from <it>Clostridium cellulovorans </it>(CBDclos) when expressed in <it>E. coli</it>. More recently we further showed that a short amphipathic helical octadecapeptide 18A (EWLKAFYEKVLEKLKELF) and a short beta structure peptide ELK16 (LELELKLKLELELKLK) have a similar property.</p> <p>Results</p> <p>In this work, we explored a third type of peptides, surfactant-like peptides, for performing such a "pulling-down" function. One or more of three such peptides (L₆KD, L₆K₂, DKL₆) were fused to the carboxyl termini of model proteins including <it>Aspergillus fumigatus </it>amadoriase II (AMA, all three peptides were used), <it>Bacillus subtilis </it>lipase A (LipA, only L₆KD was used, hereinafter the same), <it>Bacillus pumilus </it>xylosidase (XynB), and green fluorescent protein (GFP), and expressed in <it>E. coli</it>. All fusions were found to predominantly accumulate in the insoluble fractions, with specific activities ranging from 25% to 92% of the native counterparts. Transmission electron microscopic (TEM) and confocal fluorescence microscopic analyses confirmed the formation of protein aggregates in the cell. Furthermore, binding assays with amyloid-specific dyes (thioflavin T and Cong red) to the AMA-L₆KD aggregate and the TEM analysis of the aggregate following digestion with protease K suggested that the AMA-L₆KD aggregate may contain structures reminiscent of amyloids, including a fibril-like structure core.</p> <p>Conclusions</p> <p>This study shows that the surfactant-like peptides L₆KD and it derivatives can act as a pull-down handler for converting soluble proteins into active aggregates, much like 18A and ELK16. These peptide-mediated protein aggregations might have important implications for protein aggregation <it>in vivo</it>, and can be explored for production of functional biopolymers with detergent or other interfacial activities.</p

Experimental study on three-effect tubular solar still under vacuum and immersion cooling

Solar still is widely used for supplying fresh water to small communities in remote areas. One drawback of this technique lies in the low freshwater yield. Recent studies on stills of multi-effect and vacuum design proved their potential for high yield. However, such systems suffer from high electricity consumption and insufficient cooling. In this study, a novel system with a periodic pressure control scheme and water immersion cooling has been proposed to mitigate these defects. A prototype was constructed and associated with a 0.19-m2 solar panel. A 5-day outdoor experiment was conducted to evaluate the overall performance. Results indicated that the highest yield during the test was 9.8 kg/m2 at operating pressure of 40 kPa. A significant performance ratio of 1.87 was achieved with immersion cooling, i.e., 0.42 higher than that with air cooling. Thermal analysis showed that the heat transfer coefficient of water immersion cooling was 15–50 times higher than that of air cooling. Compared with previous vacuum-operated systems, the specific electricity consumption of maintaining vacuum was greatly reduced, i.e., from 21.6 kJ/kg to 1.7 kJ/kg for the case at 60 kPa. The forecast cost of the distilled water is $0.012/kg, representing an affordable desalination technique for off-grid communities

The curing characteristics and properties of bisphenol A epoxy resin/maleopimaric acid curing system

Epoxy resins are widely used as insulation materials for power systems and are mainly prepared from petrochemical materials, which have the disadvantage of being non-recyclable and environmentally harmful. In this paper, the maleopimaric acid (MPAc) curing agent was prepared from a renewable resource, rosin, and blended with a petroleum-based curing agent (methylhexahydrophthalic anhydride) to cure bisphenol A epoxy resin. The effects of the blending ratio on the curing characteristics as well as on the thermal, mechanical, and electrical properties of the epoxy resins were examined. From the results obtained, as for thermal properties, the introduction of MPAc enhanced the rigidity, glass transition temperature, and thermal decomposition temperature. As for mechanical properties, the brittleness was increased while the tensile strength and bending strength were weakened. When the mass fraction of MPAc was 20–30 wt%, the electrical properties reached the optimum, meeting the application requirements of electrical equipment. This study shows that using rosin-based curing agent maleicpine acid (MPAc) as a partial replacement for petroleum-based curing agents can make electrical equipment environmentally friendly, demonstrating the potential application prospects of rosin-based epoxy resins

Experimental investigation on an energy-efficient floor heating system with intelligent control: A case study in Chengdu, China

The space heating system accounts for 20%~50% of building energy consumption, and may lead to energy waste due to unreasonable controls. In this study, an energy-efficient floor heating system with intelligent control was proposed to improve energy efficiency of the system. In order to validate the concept of the proposed intelligent control, an experimental system was designed and constructed in Chengdu, China. Temperature, control cycle and energy consumption were then studied under different control strategies. The result shows that a larger flow rate of supply water will result in a longer control cycle and a lower control frequency, i.e., the average control cycle at 7 L min−1 is 1.7 h during the test day, while it is 1.5 h at 5 L min−1. Moreover, adopting water with a higher temperature and flow rate could achieve a higher efficiency of the system. The energy consumptions in case 1 (5 L min−1, 50 °C), case 2 (5 L min−1, 55 °C), case 5 (7 L min−1, 55 °C) and case 6 (7 L min−1, 60 °C) are 4746 kJ, 3534 kJ, 3093 kJ and 3028 kJ, respectively. Based on the experimental data, the supply water temperature is suggested to set lower than 60 °C considering human comfort

Electrical Tree Characteristics of Bisphenol A Epoxy Resin/Maleopimaric Anhydride Curing System

Epoxy resin insulation materials are mainly derived from petrochemical materials which have the disadvantages of resource consumption and environmental pollution. In order to cure bisphenol A epoxy resin, a maleopimaric anhydride (MPA) curing agent was prepared from rosin, a renewable resource, and blended with a petroleum-based curing agent (methylhexahy-drophthalic anhydride). The influence of maleopimaric anhydride content on the initiation and growth characteristics of electrical trees was studied and analyzed in this paper using molecular dynamics simulation (MD) and electrical tree tests at an 18-kilovolt power frequency voltage. When the MPA content used was &le;10%, the free volume percentage of the curing system increased with MPA content, and thus the initiation voltage became lower; when the MPA content was &ge;20%, the hydrogenated phenanthrene ring structure content increased significantly with increasing MPA content, and the rigidity of the curing system increased significantly; thus, the initiation voltage gradually increased. MPA4 had an 11.11% higher initiation voltage than the petroleum-based control group. The effect of the polar rigid structure within the curing system significantly inhibited the growth rate and length of electrical trees as MPA content increased. Electrical trees developed into light-colored, thin, and narrow dendritic structures when the MPA content reached 40%. The results show that curing epoxy resin with the rosin-based curing agent maleopimaric anhydride (MPA), in place of a petroleum-based curing agent, can produce environmentally friendly resins with excellent electrical tree resistance and potential application prospects