Microbial profile of T-shirts after a fitness session of Chinese students.

Clothing textiles could protect our human skin against external factors, but the microbial population, including conditional pathogens, in clothing, would cause unpleasant odor, Skin inflammation, and textile deterioration. Several studies have reported that microbiomes on clothes are affected by skin microorganisms of individuals, the local environment and the types of textile fabrics, but little is known about how the textile microbial community is shaped in the Chinese population. In this study, 10 healthy young students were recruited to successfully wear the T-shirts made with 3 different fabrics (polyester, cotton, and blending fabrics of polyester and cotton) during physical exercise. Total deoxyribonucleic acid (DNA) was extracted from 30 T-shirts and 16s rRNA gene amplicon sequencing was applied to estimate the absolute abundances of bacteria in the samples. The main bacteria on wore T-shirts were Staphylococcus (21.66%) Enhydrobacter (13.81%), Pantoea (8.14%), Acinetobacter (7.81%), Pseudomonas (6.18%), Cutibacterium (4.99%). However, no difference of α and ß diversity was observed among the three textile fabrics. Further analysis found that Pantoea and Pseudomonas, mainly from the environment, enriched on the polyester, but not on cotton, while Enhydrobacter, from human skin, has the growth advantage on cotton, and the blending fabric in between. Collectively, our study preliminary explored the clothes microbiome in Chinese young students, contributing to helping understand the role of clothing microorganisms on human health.

A cleavable self-assembling tag strategy for preparing proteins and peptides with an authentic N-terminus.

Recombinant protein expression and purification remains a central need for biotechnology. Herein, the authors report a streamlined protein and peptide purification strategy using short self-assembling peptides and a C-terminal cleavage intein. In this strategy, the fusion protein is first expressed as an aggregate induced by the self-assembling peptide. Upon simple separation, the target protein or peptide with an authentic N-terminus is then released in the solution by intein-mediated cleavage. Different combinations of four self-assembling peptides (ELK16, L6 KD, FK and FR) with three inteins (Sce VMA, Mtu ΔI-CM and Ssp DnaB) were explored. One protein and two peptides were used as model polypeptides to test the strategy. The intein Mtu ΔI-CM, which has pH-shift inducible cleavage, was found to work well with three self-assembling peptides (L6 KD, FR, FK). Using this intein gave a yield of protein or peptide comparable with that from other more established strategies, such as the Trx-strategy, but in a simpler and more economical way. This strategy provides a simple and efficient method by which to prepare proteins and peptides with an authentic N-terminus, which is especially effective for peptides of 30-100 amino acids in length that are typically unstable and susceptible to degradation in Escherichia coli.

Aggregating tags for column-free protein purification.

Protein purification remains a central need for biotechnology. In recent years, a class of aggregating tags has emerged, which offers a quick, cost-effective and column-free alternative for producing recombinant proteins (and also peptides) with yield and purity comparable to that of the popular His-tag. These column-free tags induce the formation of aggregates (during or after expression) when fused to a target protein or peptide, and upon separation from soluble impurities, the target protein or peptide is subsequently released via a cleavage site. In this review, we categorize these tags as follows: (i) tags that induce inactive protein aggregates in vivo; (ii) tags that induce active protein aggregates in vivo; and (iii) tags that induce soluble expression in vivo, but aggregates in vitro. The respective advantages and disadvantages of these tags are discussed, and compared to the three conventional tags (His-tag, maltose-binding protein [MBP] tag, and intein-mediated purification with a chitin-binding tag [IMPACT-CN]). While this new class of aggregating tags is promising, more systematic tests are required to further the use. It is conceivable, however, that the combination of these tags and the more traditional columns may significantly reduce the costs for resins and columns, particularly for the industrial scale.

Formation of active inclusion bodies induced by hydrophobic self-assembling peptide GFIL8.

BACKGROUND: In the last few decades, several groups have observed that proteins expressed as inclusion bodies (IBs) in bacteria could still be biologically active when terminally fused to an appropriate aggregation-prone partner such as pyruvate oxidase from Paenibacillus polymyxa (PoxB). More recently, we have demonstrated that three amphipathic self-assembling peptides, an alpha helical peptide 18A, a beta-strand peptide ELK16, and a surfactant-like peptide L6KD, have properties that induce target proteins into active IBs. We have developed an efficient protein expression and purification approach for these active IBs by introducing a self-cleavable intein molecule. RESULTS: In this study, the self-assembling peptide GFIL8 (GFILGFIL) with only hydrophobic residues was analyzed, and this peptide effectively induced the formation of cytoplasmic IBs in Escherichia coli when terminally attached to lipase A and amadoriase II. The protein aggregates in cells were confirmed by transmission electron microscopy analysis and retained ~50% of their specific activities relative to the native counterparts. We constructed an expression and separation coupled tag (ESCT) by incorporating an intein molecule, the Mxe GyrA intein. Soluble target proteins were successfully released from active IBs upon cleavage of the intein between the GFIL8 tag and the target protein, which was mediated by dithiothreitol. A variant of GFIL8, GFIL16 (GFILGFILGFILGFIL), improved the ESCT scheme by efficiently eliminating interference from the soluble intein-GFIL8 molecule. The yields of target proteins at the laboratory scale were 3.0-7.5 µg/mg wet cell pellet, which is comparable to the yields from similar ESCT constructs using 18A, ELK16, or the elastin-like peptide tag scheme. CONCLUSIONS: The all-hydrophobic self-assembling peptide GFIL8 induced the formation of active IBs in E. coli when terminally attached to target proteins. GFIL8 and its variant GFIL16 can act as a "pull-down" tag to produce purified soluble proteins with reasonable quantity and purity from active aggregates. Owing to the structural simplicity, strong hydrophobicity, and high aggregating efficiency, these peptides can be further explored for enzyme production and immobilization.

Cleavable self-aggregating tags (cSAT) for protein expression and purification.

Rapid protein expression and purification remains a critical technological need, in particular as the number of proteins being identified is exploding. In this chapter, we describe a simple and rapid scheme for expression and purification of recombinant proteins using Escherichia coli, by taking advantage of two self-aggregating peptide fusion tags 18A (EWLKAFYEKVLEKLKELF) and ELK16 (LELELKLKLELELKLK) that can drive target proteins into active protein aggregates in vivo. In practice, a target protein is fused at the N-terminus of the self-cleavable Mxe GyrA intein, which is followed by the 18A or ELK16 tag. The fusion protein is first expressed in the form of active aggregate and then separated by centrifugation upon cell lysis. Subsequently, the DTT-mediated intein self-cleavage reaction releases the target protein into solution. These cleavable self-aggregating tags (cSAT, intein-18A/ELK16) provide a quick and efficient route for the production of proteins with modest purity (around 90% in the case of intein-ELK16). Two application examples are included in the chapter.

Rehabilitation for the management of knee osteoarthritis using comprehensive traditional Chinese medicine in community health centers: study protocol for a randomized controlled trial.

BACKGROUND: It is becoming increasingly necessary for community health centers to make rehabilitation services available to patients with osteoarthritis of the knee. However, for a number of reasons, including a lack of expertise, the small size of community health centers and the availability of only simple medical equipment, conventional rehabilitation therapy has not been widely used in China. Consequently, most patients with knee osteoarthritis seek treatment in high-grade hospitals. However, many patients cannot manage the techniques that they were taught in the hospital. Methods such as acupuncture, tuina, Chinese medical herb fumigation-washing and t'ai chi are easy to do and have been reported to have curative effects in those with knee osteoarthritis. To date, there have been no randomized controlled trials validating comprehensive traditional Chinese medicine for the rehabilitation of knee osteoarthritis in a community health center. Furthermore, there is no standard rehabilitation protocol using traditional Chinese medicine for knee osteoarthritis. The aim of the current study is to develop a comprehensive rehabilitation protocol using traditional Chinese medicine for the management of knee osteoarthritis in a community health center. METHOD/DESIGN: This will be a randomized controlled clinical trial with blinded assessment. There will be a 4-week intervention utilizing rehabilitation protocols from traditional Chinese medicine and conventional therapy. Follow-up will be conducted for a period of 12 weeks. A total of 722 participants with knee osteoarthritis will be recruited. Participants will be randomly divided into two groups: experimental and control. Primary outcomes will include range of motion, girth measurement, the visual analogue scale, and results from the manual muscle, six-minute walking and stair-climbing tests. Secondary outcomes will include average daily consumption of pain medication, ability to perform daily tasks and health-related quality-of-life assessments. Other outcomes will include rate of adverse events and economic effects. Relative cost-effectiveness will be determined from health service usage and outcome data. DISCUSSION: The primary aim of this trial is to develop a standard protocol for traditional Chinese medicine, which can be adopted by community health centers in China and worldwide, for the rehabilitation of patients with knee osteoarthritis. CLINICAL TRIALS REGISTRATION: ChiCTR-TRC-12002538.

Facile expression and purification of the antimicrobial peptide histatin 1 with a cleavable self-aggregating tag (cSAT) in Escherichia coli.

Human histatin 1 (Hst1), a member of the histatin family, possesses antimicrobial properties. In this study, we applied a previously developed cleavable self-aggregating tag (cSAT) for the expression and purification of histatin 1 to demonstrate its utility for peptide expression and purification. The tag consists of a self-cleavable intein and a self-assembling peptide ELK16 (I-ELK16). First, an active insoluble aggregate of the recombinant histatin 1-Mxe GyrA intein-ELK16 (Hst1-I-ELK16) fusion protein was produced with a yield of 28.9 µg/mg wet cell pellet. The thiol reagent dithiothreitol (DTT) was then used to induce the intein-mediated cleavage and peptide release into the soluble fraction with a yield of 2.06 µg/mg wet cell pellet and a purity of 70%. The peptide was further purified by high performance liquid chromatography. These results were comparable to the yield and purity achieved when the more conventional glutathione transferase (GST) tag was used. The antimicrobial activities of this recombinant histatin 1 were confirmed against three Candida strains. This cSAT technique offers considerable advantages in terms of its simplicity and speed, eliminating the need for an exogenous protease, and reducing the number of chromatography purification steps. This technique should also be useful for the expression and purification of other AMPs.

Self-assembling amphipathic alpha-helical peptides induce the formation of active protein aggregates in vivo.

We recently found that several self-assembling alpha, beta, or surfactant-like peptides, when terminally attached to proteins, can promote the in vivo assembly of active protein aggregates (or active inclusion bodies, AIBs) in Escherichia coil. In this work, we systematically examined the AIBs induced by an amphipathic alpha-helical peptide 18Awt (EWLKAFYEKVLEKLKELF) and its variants with altered ion pairs. Transmission electron microscopic and Fourier transform infrared spectroscopic analyses suggested that the AIBs appeared to adopt an amorphous mesh-like structure, and were likely induced by helical structures formed by the assembly of the 18A peptides. Confocal fluorescent micrographic analysis revealed that the AIBs resided around the periphery of the cell membrane or in the cytoplasm, depending on the distribution of ion pairs on the 18A peptides, which suggested that the association between the aggregates and the cell membrane was mediated by the lipid-18A interaction. Two of these 18A peptide variants were further used in constructing cleavable self-aggregating tags (cSAT) in conjunction with an intein molecule for protein purification, and verified using two model proteins. This extends the cSAT approach for laboratory and potentially industrial uses. Our study might also provide new insights into aggregation-related diseases.

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

BACKGROUND: Inactive 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. RESULTS: 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 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. CONCLUSIONS: 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 in vivo, and can be explored for production of functional biopolymers with detergent or other interfacial activities.

Streamlined protein expression and purification using cleavable self-aggregating tags.

BACKGROUND: 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 Escherichia coli (E. coli), 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. RESULTS: 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 ß-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. CONCLUSIONS: 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.

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

BACKGROUND: 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, ß-galactosidase, ß-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 ß-amyloid peptide Aß42(F19D). As active IBs may have many attractive advantages in enzyme production and industrial applications, it is of considerable interest to explore them further. RESULTS: In 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, ß-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. CONCLUSIONS: This has been the first report where a terminally attached self-assembling ß 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.