Affinity purification of recombinant proteins is an essential technique in biotechnology. However, current affinity purification methods are very cost-intensive, and this imposes limits on versatile use of affinity purification for obtaining purified proteins for a variety of applications. To overcome this problem, we developed a new affinity purification system which we call CSAP (chitin- and streptavidin-mediated affinity purification) for low-cost purification of Strep-tagâ II fusion proteins. The CSAP system is designed to utilize commercially available chitin powder as a chromatography matrix, thereby significantly improving the cost-efficiency of protein affinity purification. We investigated the CSAP system for protein screening in 96-well format as a demonstration. Through the screening of 96â types of purified hemoproteins, several proteins capable of the catalytic diastereodivergent synthesis of cyclopropanes were identified as candidates for an abiotic carbene transfer reaction.
Chitin , Escherichia coli , Streptavidin/chemistry , Chitin/chemistry , Escherichia coli/metabolism , Recombinant Proteins/chemistry , Chromatography, Affinity/methods , Recombinant Fusion Proteins/chemistry
Enzymes inherently exhibit molecule-to-molecule heterogeneity in their conformational and functional states, which is considered to be a key to the evolution of new functions. Single-molecule enzyme assays enable us to directly observe such multiple functional states or functional substates. Here, we quantitatively analyzed functional substates in the wild-type and 69 single-point mutants of Escherichia coli alkaline phosphatase by employing a high-throughput single-molecule assay with a femtoliter reactor array device. Interestingly, many mutant enzymes exhibited significantly heterogeneous functional substates with various types, while the wild-type enzyme showed a highly homogeneous substate. We identified a correlation between the degree of functional substates and the level of improvement in promiscuous activities. Our work provides much comprehensive evidence that the functional substates can be easily altered by mutations, and the evolution toward a new catalytic activity may involve the modulation of the functional substates.
Alkaline Phosphatase , Escherichia coli Proteins , Escherichia coli , Protein Conformation , Alkaline Phosphatase/chemistry , Alkaline Phosphatase/genetics , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Mutation
We isolated four Parageobacillus strains from soil in Japan and completely sequenced their genomes. Three of four strains showed ≥98.9% average nucleotide identity (ANI) to Parageobacillus caldoxylosilyticus S1812T, while one strain, designated KH3-4, showed the highest ANI (91%) to Parageobacillus thermantarcticus M1T, suggesting the species novelty of KH3-4.
Extremely thermophilic strains belonging to the genus Thermus were isolated from Senami Hot Spring in Japan. Here, I report the complete genome sequences of five Thermus thermophilus strains and one Thermus brockianus strain, which were obtained by combining Oxford Nanopore long-read and DNBSEQ or Illumina short-read sequencing data.
Complete genome resequencing was conducted for Thermus thermophilus strain TMY by hybrid assembly of Oxford Nanopore Technologies long-read and MGI short-read data. Errors in the previously reported genome sequence determined by PacBio technology alone were corrected, allowing for high-quality comparative genomic analysis of closely related T. thermophilus genomes.
We isolated four Thermus thermophilus strains from Arima Hot Spring in Japan. Complete genome sequencing revealed that they showed average nucleotide identities of ≥99.21% to each other and to strains previously isolated from the same spot, but of ≤97.86% to strains from geographically different spots in Japan, reflecting habitat-specific genomic conservation.
We isolated Thermus thermophilus strains HB5002 and HB5008 from Mine Hot Spring in Japan. Whole-genome sequencing revealed that they showed â¼100% average nucleotide identity to each other, ≥98.53% to the T. thermophilus strains originating from the same spot but ≤97.64% to the T. thermophilus strains from geographically different places in Japan.
We isolated Thermus thermophilus strain HB5018 from Mine Hot Spring in Japan, where the type strain HB8 was isolated nearly half a century ago. The complete genome sequence of HB5018 showed 99.1% average nucleotide identity with HB8, suggesting strict species conservation in the habitat over the past 50 years.
A thiocyanate-degrading bacterium, Thiohalobacter sp. strain COW1, was isolated from activated sludge treating coke oven wastewater, and the complete genome sequence was determined. COW1 contained a single circular chromosome (3.23 Mb; G+C content, 63.4%) in which 2,788 protein-coding genes, 39 tRNA genes, and 3 rRNA genes were identified.
IMPORTANCE: High placebo response can often mask the evaluation of active treatment in clinical studies for women with hot flashes and potentially undermine the evaluation of new treatments. OBJECTIVE: The aim of this meta-analysis was to determine the factors associated with high placebo response (defined as the reduction in the mean number of hot flash frequency from baseline) in randomized, controlled, double-blind studies enrolling women with hot flashes. EVIDENCE REVIEW: To identify eligible studies, Embase, MEDLINE, and BIOSIS Previews were searched for English-language articles published between April 1975 and August 2020. Placebo-controlled, double-blind, randomized studies that assessed changes in hot flash frequency were included if they satisfied the defined criteria. We conducted univariate and multivariate analyses using categorical and numerical data. Categorical data included the following variables and levels in brackets: active treatment type (hormone therapy /non- hormone therapy /complementary and alternative medicine), administration route (oral/non-oral), study region (in/excluded the US), breast cancer population (in/excluded), entry criteria of hot flash severity (moderate to severe only/all included), parallel or crossover study, placebo run-in period before treatment (yes/no), and menopausal status (postmenopausal only/include perimenopausal/include premenopausal). Numerical data included published year, pretreatment period duration, treatment period duration, number of sites, number of total participants, number of placebo participants, number of treatment arms, mean age, BMI, and hot flash frequency at baseline. FINDINGS: Forty-three of the 802 identified publications were included in the review. Multivariate analysis identified three individual factors associated with high placebo response: treatment period duration, number of treatment arms, and BMI. CONCLUSIONS AND RELEVANCE: We identified several factors associated with high placebo response in clinical studies of women with hot flashes. Knowing these factors may enable proactive implementation of operational and analytic strategies that further aid in determining the true treatment effect of an intervention.
Hot Flashes , Placebo Effect , Cross-Over Studies , Double-Blind Method , Female , Hot Flashes/drug therapy , Humans , Randomized Controlled Trials as Topic , Treatment Outcome
We report here the complete genome sequence of Geobacillus sp. strain E55-1, isolated from the hot sediments of Mine Geyser in Japan. This strain exhibited â¼85% average nucleotide identity and â¼98.5% 16S rRNA sequence identity with the most closely related Geobacillus species.
We isolated the novel strain Vibrio rotiferianus AM7 from the shell of an abalone. In this article, we report the complete genome sequence of this organism, which was obtained by combining Oxford Nanopore long-read and Illumina short-read sequencing data.
We isolated Pseudomonas otitidis strain MrB4 from the near-shore area of Lake Biwa in Japan and generated its complete genome sequence. MrB4 possesses a single circular chromosome of 6,089,454 bp, with â¼97% average nucleotide identity to the P. otitidis type strain MCC10330 (draft genome).
We isolated the soil bacterium strain PL1 and herein report its complete genome sequence. The strain presented 97% average nucleotide identity (ANI) to Bacillus cereus and 91% ANIs to other members of the B. cereus group, indicating that it is affiliated with B. cereus.
We isolated Rhodothermus marinus strains AA2-13 and AA3-38 from Arima Onsen, a hot spring in Japan, and sequenced their genomes. The average nucleotide identity between their genomes was 99.2%, and that with the genome of R. marinus strain DSM 4252T (isolated from Iceland) was â¼95.2%, suggesting close relationships among these strains.
A novel strain of Alteromonas, I4, was isolated from a shallow beach of the Japan Sea. Here, we report the complete genome sequence of I4; this strain contains a single circular chromosome (5,133,645 bp; G+C content, 48.4%) and a single circular putative plasmid (123,836 bp; G+C content, 45.1%).
Staphylococcus arlettae is one coagulase-negative species in the bacterial genus Staphylococcus Here, we describe the closed complete genome sequence of S. arlettae strain P2, which was obtained using a hybrid approach combining Oxford Nanopore long-read and Illumina MiSeq short-read sequencing data.
Thermus thermophilus strain HC11 was isolated from Mine Geyser in Japan, where type strain HB8 was isolated 50 years ago. In this article, the complete genome sequence of HC11 is presented. HC11 shares the highest average nucleotide identity with HB8 among known T. thermophilus genomes (93.1%) with no genetic rearrangements.
Rubrobacter xylanophilus strain AA3-22, belonging to the phylum Actinobacteria, was isolated from nonvolcanic Arima Onsen (hot spring) in Japan. Here, we report the complete genome sequence of this organism, which was obtained by combining Oxford Nanopore long-read and Illumina short-read sequencing data.
We report a general strategy based on digital counting principle that enables an efficient acquisition of enzyme mutants with desired activities from just a few clones within a day. We prepared a high-density femtoliter droplet array, consisting of 1 million uniform droplets per 1 cm2 to carry out high-throughput protein synthesis and screening. Single DNA molecules were randomly distributed into each droplet following a Poisson process to initiate the protein synthesis with coupled cell-free transcription and translation reactions and then recovered by a microcapillary. The protein yield in each droplet was proportional to the number of DNA molecules, meaning that droplets with apparent intensities higher than the Poisson distribution-predicted maximum can be readily identified as the exact hits exhibiting the desired increased activity. We improved the activity of an alkaline phosphatase up to near 20-fold by using less than 10 nl of reagents.
Biosensing Techniques , High-Throughput Screening Assays , Microfluidic Analytical Techniques , Algorithms , DNA, Single-Stranded/analysis , Models, Theoretical , Proteins/analysis , Reproducibility of Results
