BackgroundIn 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, D-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.ResultsIn this paper, we report that an ionic self-assembling peptide ELK16 (LELELKLK)2 was able to effectively induce the formation of cytoplasmic inclusion bodies in Escherichia coli (E. coli) 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.ConclusionsThis 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 E. coli. It has the potential to render E. coli and other recombinant hosts more efficient as microbial cell factories for protein production. Our observation might also provide hints for protein aggregation-related diseases.

BackgroundInactive protein inclusion bodies occur commonly in Escherichia coli (E. coli) 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 Clostridium cellulovorans (CBDclos) when expressed in E. coli. 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.ResultsIn 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 Aspergillus fumigatus amadoriase II (AMA, all three peptides were used), Bacillus subtilis lipase A (LipA, only L6KD was used, hereinafter the same), Bacillus pumilus xylosidase (XynB), and green fluorescent protein (GFP), and expressed in E. coli. 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.ConclusionsThis 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 in vivo, and can be explored for production of functional biopolymers with detergent or other interfacial activities.

Amyloid fibrillation of proteins is a hallmark of neurodegenerative disease, accompanied by the formation of the organized cross‐β cores. This conformational transformation is considered to be related to the toxicity underlying the pathogenic mechanism. However, the exact conformational transformation kinetics of amyloid fibrillation are not fully understood. Herein, Raman spectroscopy was used to detect the transformation in the molecular structure of hen egg white lysozyme during amyloid formation under heat and acidic conditions (pH 2.0 and 65°C). The overall kinetics of the hen egg white lysozyme conformational change were investigated by analyzing five characteristic spectral fingerprints. The two N–Cα–C stretching bands at 899 and 935 cm−1 and the amide I band (at 1,640–1,680 cm−1) correlated to the lysozyme skeleton structure, whereas the band of the Phe amino acid group in side chains at 1,003 cm−1 and the two Trp residue bands at 760 and 1,340–1,360 cm−1 were associated with the tertiary structure. Based on these results, a four‐stage step‐by‐step transformation mechanism is first proposed to describe the exact kinetics of lysozyme amyloid fibrillation under heat and acidic conditions. This provides necessary information for physiologists to artificially control the amyloid formation of neurodegenerative disease patients.

A novel stabilized hemocompatible multicomponent coating was engineered by consecutive alternating adsorption of two polysaccharides, alginate (Alg) and heparin (Hep), onto a Nitinol surface via electrostatic interaction in combination with photoreaction in situ. For this purpose, a photosensitive cross-linker, p-diazonium diphenyl amine polymer (PA), was used as an interlayer between alginate and heparin. The optical intensity of UV/vis spectra increased linearly with the number of layers, indicating the buildup of a multilayer structure and uniform coating. Photo-cross-linking resulted in higher stability without compromising its catalytic capacity to promote antithrombin III (ATIII)-mediated thrombin inactivation. Chromogenic assays for heparin activity proved definitively that anticoagulation activity really comes from surface-bound heparin in multilayer film, not from solution-phase free heparin that has leaked from multilayer film. The activated partial thromboplastin time (aPTT) assay showed that both (PA/Hep)8- and (PA/Alg/PA/Hep)4-coated Nitinol were less thrombogenic than the uncoated one. Yet, the latter was found to be more stable under a continuous shaken wash. In addition, (PA/Alg/PA/Hep)4 film exhibited lower surface roughness and higher hydrophilicity than (PA/Hep)8. As a result, hemolysis of (PA/Alg/PA/Hep)4 (0.34 +/- 0.064%) was lower than (PA/Hep)8 (0.52 +/- 0.241%). The naked Nitinol and (PA/Hep)8-coated Nitinol showed relatively strong platelet adhesion. On the contrary, no sign of any cellular matter was seen on the (PA/Alg/PA/Hep)4 surface. It is believed that the phenomenon of interlayer diffusion resulted in blended structures, hence, the enhanced wettability and antifouling properties after the incorporation of alginate layers. It is likely that the cooperative effect of alginate and heparin led to the excellent blood compatibility of the (PA/Alg/PA/Hep)4 coating. To simplify, there is greater advantage in utilizing cross-linked alginate/heparin surfaces rather than merely the heparin surface for improving blood- and tissue-compatible devices.