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

Composite Edible Film Containing Microcapsules Composed of Shrimp-derived Bioactive Peptide Preparation and Preservation Potential

Shrimp body had to face with spoilage and melanosis when it come to food preservation. In this study, a kind of composite edible film (CEF) composed of hydroxypropyl methyl cellulose (HPMC) and shrimp-derived bioactive peptide microcapsule (SBPM), was developed to preserve the body of Penaeus vannamei. First of all, single-factor experiments were conducted by monitoring the mechanical performance and the water vapor permeability (WVP) of CEF, in order to investigate the influence of the dosages of HPMC, glycerol and SBPM, upon the formulation properties and mechanical strength of the film. The FTIR and free radical scavenging capacity of CEF were also determined when the SBPM addition changed and then, the optimum preparation process of CEF was ascertained based on the results mentioned above. In addition, the preservation-effect of CEF on fresh Penaeus vannamei was evaluated by inspecting some important items such as the sensory evaluation, pH, total volatile basic nitrogen (TVB-N) and aerobic bacterial count of shrimp flesh. The results of the single-factor experiments indicated the optimum amounts of HPMC, glycerol and SBPM were 12%, 0.4% and 3%, respectively, and the comprehensive performance of CEF was satisfied under this optimum condition. The antioxidant ability of CEF was excellent when the dosage of SBPM was not less than 3% and, the interaction between SBPM and other membrane fractions was powerful when the addition amount of SBPM came to be 3%~4%, which was indicated by the FTIR results. More importantly, the spoilage of shrimp body could be controlled by covering the body with CEF and, the sensory of seafood might be maintained by this film, as evidenced by the inhibitory effects of CEF on the raising of body pH, the jump of TVB-N and the increase of aerobic bacterial count in shrimp fresh. The quality guarantee period of fresh body in CEF group could be prolonged by 3~4 days as compared with that in unprotected group, while the overall freshness-keeping ablility of CEF was superior to that of HPMC film even PE film, indicating the potent protective effect of CEF on the fresh of Penaeus vannamei

Cloning and Expression of Aspergillus tamarii FS132 Lipase Gene in Pichia pastoris

A lipase gene (atl) was cloned from Aspergillus tamarii FS132 for the first time. The gene was found to have an open reading frame of 1024 base pairs (bp), and the coding region of the gene contained two introns (51 bp and 52 bp). Multi-alignment analysis of the deduced amino acid sequence indicated high homology between the enzyme and mono-and diacylglycerol lipases from fungi Aspergillus. The recombinant lipase was expressed in Pichia pastoris GS115 cells. The recombinant lipase was found to have a molecular mass of 36.7 kDa, and it exhibited lipase activity of 20 U/mL in culture supernatant when tributyrin was used as the substrate

Pre‐symptomatic transmission of novel coronavirus in community settings

We used contact tracing to document how COVID‐19 was transmitted across 5 generations involving 10 cases, starting with an individual who became ill on January 27. We calculated the incubation period of the cases as the interval between infection and development of symptoms. The median incubation period was 6.0 days (interquartile range, 3.5‐9.5 days). The last two generations were infected in public places, 3 and 4 days prior to the onset of illness in their infectors. Both had certain underlying conditions and comorbidity. Further identification of how individuals transmit prior to being symptomatic will have important consequences.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/163478/2/irv12773.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163478/1/irv12773_am.pd

Research progress of nitrogen removal by anaerobic ammoxidation combined process

Anaerobic ammonia oxidation (ANAMMOX) is an efficient and low-fare biological nitrogen removal process. Nevertheless, there are still some challenges in mainstream request due to the limitations of substrate type and nitrate accumulation. At the present, anaerobic ammox combined process has been widely studied to solve the above issue. In this paper, the anammox combined processes developed in recent years are reviewed, and the process principles, pros and cons, influencing factors, process scalability, key bottlenecks in application and relevant work of the investigation group are discussed. Finally, the evolution of complex anammox process in urban domestic wastewater treatment was prospected

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