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

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

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

Novel Ternary Absorbent: Dibutylamine Aqueous–Organic Solution for CO₂ Capture

Amine aqueous–organic solution is a novel ternary absorbent for CO₂ capture. In the present work, 0.5 mol L^–1 dibutylamine (DBA)/water/ethanol was chosen as the ternary mixed absorbent to obtain high absorption capacity and regeneration efficiency. The results demonstrated that the DBA/water/ethanol solution took into account the advantages of aqueous and non-aqueous solutions. When the volume ratio of water and ethanol was 5:5 at 313 K, the absorption loading reached a maximum of 0.82 mol mol^–1, which was higher than that of DBA/ethanol or DBA/water, and the viscosity of saturated solution was only 1.72 mPa s. Meanwhile, the regeneration efficiency of saturated DBA/water/ethanol solution reached 97.6% at 373 K for 30 min and still obtained 94.3% after the fifth regeneration cycle. Moreover, the detailed absorption/desorption mechanisms could be explained by carbon-13 nuclear magnetic resonance that DBA initially reacted with CO₂ to form carbamate and then carbamate further reacted with water and ethanol to form HCO₃^– and C₂H₅OCO₂^–. During the absorption process, HCO₃^– and C₂H₅OCO₂^– could be converted into each other in the water/ethanol mixed solvent and finally reached equilibrium. In addition, the high regeneration efficiency of the DBA/water/ethanol solution was attributed to the reason that all carbamate converted to carbonates of HCO₃^– and C₂H₅OCO₂^– in the saturated solution and HCO₃^– and C₂H₅OCO₂^– decomposed more easily than carbamate during desorption

Significance of Prehistoric Liquefaction Features in the Xilinhot District, Inner Mongolia, Northern China

Paleo-ground ruptures, fissures, liquefaction and geomorphic features in the Xilinhot district, Inner Mongolia, northern China, are documented and seismic intensity for the meizoseismal areas and magnitudes of paleoearthquakes are estimated. Trenching investigations revealed a huge paleoseismic ground rupture and fissure zone with a width of more than 200 m. Field investigations and interpretation of remote sensing images demonstrate that active faults related to paleoearthquakes appear to be about 200 km long. Geologic and geomorphic evidences indicate that one large earthquake with a magnitude of about 7.5 at about 13 ka BP or multiple paleoseismic events of M ¡_ 6.0 have occurred in the studied area since 53 ka BP. One of the paleo-meizoseismal areas is determined to be near Xilinhot. Seismic intensity in Modified Mercalli scale (MM) is estimated to be larger than VI in the vicinity of the study area and at least VIII in the epicentral region. This is consistent with seismic activities in and around the Xilinhot district in recent years, but higher than anything yet reported. The results provide important data for design engineering and regional planning in order to resist damage from potentially large earthquakes in the future