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Procalcitonin (PCT) (i.e. a precursor of calcitonin) attracts much attention as a reliable biomarker of bacterial infections because its concentration increases rapidly in the blood when bacterial infections occur in the body. Sepsis may occur due to indiscriminate and vigorous proliferation of infectious bacteria, and accordingly early diagnosis and treatment of bacterial infection are of crucial importance. However, current diagnostic methods for sepsis suffer from long assay time, multiple and complex assay steps, inaccuracy, and requirement of analytical equipments. The goal of this study is to develop an advanced one-step-immunoassay that enables quick and accurate diagnosis of sepsis through measuring the PCT concentration in patient sera, which is based on self-enhancement of optical detection signals from large gold particles (i.e. clusters of gold nanoparticles) that are formed on the agglomerates of PCT-bound 3-dimensional (3D) probes. The 3D probe is constructed through attaching polyclonal anti-PCT antibodies (IgGs) to the surface of a modified hepatitis B virus (HBV) capsid, where both tandem repeats of the B domain of Staphylococcal protein A (SPAB) and the hexa-histidine tag are inserted into each HBV core protein (i.e. subunit of HBV capsid). That is, anti-PCT IgGs are attached via strong interaction between the Fc region and surface-exposed SPAB. Furthermore, hook effect-free and PCT concentration-dependent optical signals were consistently generated by adding both bovine serum albumin (BSA) and nickel ions to patient sera and also by optimally adjusting the 3D probe concentration. Compared to conventional chemiluminescent microparticle immunoassay (CMIA) showing poor linearity of detection signals, this novel immunoassay accurately detected PCT with good linearity between PCT concentrations and optical signals in a wide range of PCT concentrations (0.05–200 ng mL−1) and also showed a sufficiently low limit of detection, resulting in 100% sensitivity and 100% specificity when tested with 30 sepsis patients and 30 healthy individuals.

Proteins are fundamentally the most important macromolecules for biochemical, mechanical, and structural functions in living organisms. Therefore, they provide us with diverse structural building blocks for constructing various types of biomaterials, including an important class of such materials, hydrogels. Since natural peptides and proteins are biocompatible and biodegradable, they have features advantageous for their use as the building blocks of hydrogels for biomedical applications. They display constitutional and mechanical similarities with the native extracellular matrix (ECM), and can be easily bio-functionalized via genetic and chemical engineering with features such as bio-recognition, specific stimulus-reactivity, and controlled degradation. This review aims to give an overview of hydrogels made up of recombinant proteins or synthetic peptides as the structural elements building the polymer network. A wide variety of hydrogels composed of protein or peptide building blocks with different origins and compositions - including β-hairpin peptides, α-helical coiled coil peptides, elastin-like peptides, silk fibroin, and resilin - have been designed to date. In this review, the structures and characteristics of these natural proteins and peptides, with each of their gelation mechanisms, and the physical, chemical, and mechanical properties as well as biocompatibility of the resulting hydrogels are described. In addition, this review discusses the potential of using protein- or peptide-based hydrogels in the field of biomedical sciences, especially tissue engineering.

As recent advancements in the chimeric antigen receptor-T cells have revolutionized the way blood cancers are handled, potential benefits from producing off-the-shelf, standardized immune cells entail the need for development of allogeneic immune cell therapy. However, host rejection driven by HLA disparity in adoptively transferred allogeneic T cells remains a key obstacle to the universal donor T cell therapy. To evade donor HLA-mediated immune rejection, we attempted to eliminate T cell’s HLA through the CRISPR/Cas9 gene editing system. First, we screened 60 gRNAs targeting B2M and multiple sets of gRNA each targeting α chains of HLA-II (DPA, DQA and DRA, respectively) using web-based design tools, and identified specific gRNA sequences highly efficient for target deletion without carrying off-target effects. Multiplex genome editing of primary human T cells achieved by the newly discovered gRNAs yielded HLA-I- or HLA-I/II-deficient T cells that were phenotypically unaltered and functionally intact. The overnight mixed lymphocyte reactions demonstrated the HLA-I-negative cells induced decreased production of IFN-γ and TNF-α in alloreactive T cells, and deficiency of HLA-I/II in T cells further dampened the inflammatory responses. Taken together, our approach will provide an efficacious pathway toward the universal donor cell generation by manipulating HLA expression in therapeutic T cells.

Since the coronavirus disease outbreak in 2019, several antibody therapeutics have been developed to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Antibody therapeutics are effective in neutralizing the virus and reducing hospitalization in patients with mild and moderate infections. These therapeutics target the spike protein of SARS-CoV-2; however, emerging mutations in this protein reduce their efficiency. In this study, we developed a universal SARS-CoV-2 neutralizing antibody. We generated a humanized monoclonal antibody, MG1141A, against the receptor-binding domain of the spike protein through traditional mouse immunization. We confirmed that MG1141A could effectively neutralize live viruses, with an EC50 of 92 pM, and that it exhibited effective Fc-mediated functions. Additionally, it retained its neutralizing activity against the alpha (UK), beta (South Africa), and gamma (Brazil) variants of SARS-CoV-2. Taken together, our study contributes to the development of a novel antibody therapeutic approach, which can effectively combat emerging SARS-CoV-2 mutations.

Methane monooxygenase (MMO), which exists in particulate (pMMO) or soluble forms (sMMO) in methanotrophic bacteria, is an industrially promising enzyme that catalyses oxidation of low-reactive methane and other carbon feedstocks into methanol and their corresponding oxidation products. However, the simple, fast and high-yield production of functionally active MMO, which has so far been unsuccessful despite diverse approaches based on either native methanotroph culture or recombinant expression systems, remains a major challenge for its industrial applications. Here we developed pMMO-mimetic catalytic protein constructs by genetically encoding the beneficial reassembly of catalytic domains of pMMO on apoferritin as a biosynthetic scaffold. This approach resulted in high-yield synthesis of stable and soluble protein constructs in Escherichia coli, which successfully retain enzymatic activity for methanol production with a turnover number comparable to that of native pMMO. Methane monooxygenase (MMO)—a protein of high biotechnological interest for the selective and mild conversion of methane to methanol—lacks a high-throughput production system. Now, the authors report the efficient production of particulate MMO-mimics for catalysis in solution and hydrogels.

This study developed a new method for forensic saliva identification using three oral bacteria, Streptococcus salivarius, Streptococcus sanguinis, and Neisseria subflava, combined with a real-time polymerase chain reaction (RT-PCR) system we called OB mRT-PCR. Analytical sensitivity results showed that the target bacteria were amplified at 102–107 copies/reaction, and analytical specificity was assessed using 24 other viruses, bacteria, and protozoa. To evaluate the OB mRT-PCR kit for forensic applications, saliva from 140 Korean individuals was tested, and at least two target bacteria were detected in all the samples. Additional studies on non-saliva samples demonstrated the specificity of the kit. Comparison of the kit with two conventional saliva test methods, the SALIgAE and RSID-Saliva assays, indicated that it was more sensitive and applicable to saliva samples in long-term storage (up to 14 weeks). Additionally, through amplification of mock forensic items and old DNA samples (isolated without lysis of the bacterial cells, regardless of their Gram-positivity), we found that the kit was applicable to not only saliva swabs, but also DNA samples. We suggest that this simple RT-PCR-based experimental method is feasible for rapid on-site analysis, and we expect this kit to be useful for saliva detection in old forensic DNA samples.

The endocytosis-mediating performances of two types of peptide ligands, cell receptor binding peptide (CRBP) and cell membrane penetrating peptide (CMPP), were analyzed and compared using a common carrier of peptide ligands-human ferritin heavy chain (hFTH) nanoparticle. Twenty-four copies of a CMPP(human immunodeficiency virus-derived TAT peptide) and/or a CRBP (peptide ligand with strong and specific affinity for either human integrin(αv β3 ) or epidermal growth factor receptor I (EGFR) that is overexpressed on various cancer cells) were genetically presented on the surface of each hFTH nanopariticle. The quantitative level of endocytosis and intracellular localization of fluorescence dye-labeled CRBP- and CMPP-presenting nanoparticles were estimated in the in vitro cultures of integrin- and EGFR-overexpressing cancer and human dermal fibroblast cells(control). From the cancer cell cultures treated with the CMPP- and CRBP-presenting nanoparticles, it was notable that CRBPs resulted in quantitatively higher level of endocytosis than CMPP (TAT) and successfully transported the nanoparticles to the cytosol of cancer cells depending on concentration and treatment period of time, whereas TAT-mediated endocytosis localized most of the nanoparticles within endosomal vesicles under the same conditions. These novel findings provide highly useful informations to many researchers both in academia and in industry who are interested in developing anticancer drug delivery systems/carriers.

In this paper, we describe the development of a novel method to detect oral bacteria by combining direct polymerase chain reaction (direct PCR) with an immunochromatographic strip (ICS), enabling the identification of saliva in forensic samples. Direct PCR was first used to directly amplify specific oral bacterial sequences (from Streptococcus sanguinis and Streptococcus salivarius) from swab samples, circumventing the need for tedious sample preparation steps such as cell lysis and DNA extraction and purification. The resultant amplicons were then colorimetrically detected on an ICS, a much more convenient, cost-effective, and user-friendly detection method than those currently available, thereby allowing the presence or absence of the target oral bacteria to be determined with the naked eye. Moreover, the entire analysis process was performed rapidly and with ease using this combination of direct PCR amplification from swab samples and ICS-based amplicon detection. This method successfully detected S. sanguinis and S. salivarius in most of the saliva swab samples tested, and returned negative results using blood, semen, urine, and vaginal fluid swab samples. Furthermore, S. sanguinis and S. salivarius were detected in a large number of mock forensic samples using this technique, which suggests that direct PCR and ICS-based detection of oral bacteria is sufficient to demonstrate the presence of saliva. Thus, we believe that the proposed method could be very useful for the identification of saliva in forensic applications.

The objective of this study is to develop an accurate, rapid, simple, and label-free assay technology that enables point-of-care diagnosis of AIDS. For this, 3-dimensional (3D) probes to sensitively detect anti-HIV antibodies were designed and synthesized by genetically presenting a HIV antigen (gp41) on the surface of engineered human ferritin nanoparticles. The 3D probes also present multi-copies of the hexa-histidine peptide (H6) on their surface to chemisorb gold ions (Au3+), which is essential for the generation and self-enhancement of assay signals. The developed new assay technology (named “one-step-immunoassay”) quickly produced clear optical signals through a simple and convenient one-step procedure. The diagnostic performance of the one-step-immunoassay was compared with that of the conventional lateral flow assay (LFA) using 30 AIDS patient and 20 healthy sera. The sensitivity of LFA was only 63% when a single antigen (gp41) was used but enhanced to 90% when three different antigens (gp41, p24, and gp120) were used together as the assay probes. In contrast, the one-step-immunoassay using only gp41 produced strong optical signals within 15 min without causing any false negative/positive signals, showing 100% sensitivity and 100% specificity and holding promising potential for clinical point-of-care diagnosis of AIDS.

Recently, a method of identifying body fluids using DNA methylation has been developed (Frumkin et al., 2011). An existing multiplex assay using 9 CpG markers could differentiate 5 body fluids: semen, blood, saliva, menstrual blood, and vaginal fluid. To validate this technique, we evaluated the previously described body fluid identification method by means of single base extension (SBE). DNA methylation was applied to 22 samples in 18 forensic cases; seven of these were semen, three were blood, eight were saliva, three were vaginal fluid, and one was menstrual blood. Total of 18 samples were tested, the DNA methylation profiles were coincident from preliminary tests (acid phosphatase (AP), leucomalachite green (LMG, Sigma Aldrich, St Louis, MO, USA) and SALIgAE®) except one sample which displayed an all-negative result. After applying the DNA methylation method to forensic samples, we determined that it could be very useful for differentiating vaginal secretions from menstrual blood, for which there is no conventional preliminary testing method.

The aim of this study was to develop the economic and effective buffer system for microalgae mass cultivation using industrial flue gas. Due to the continuous flue gas supplement, culture media acidified, therefore cell growth inhibited. Although buffering agent was added, this result increase in cost for overall culture process. Therefore combined buffer system of bicarbonate and phosphate (BP) for large-scale use was investigated. The bicarbonate buffer system generated from CO2 dissolution, additionally phosphate buffer system improves the buffer performance under the continuous CO2 supplementation from flue gas. The microalgae Haematococcus pluvialis was cultivated under autotrophic outdoor conditions using these buffer solutions. As a result, the autotrophic BP buffer system enhanced the biomass and astaxanthin productivity of H. pluvialis to 105% and 103%, respectively. The results confirm that the BP buffer system reduces the cost of microalgal CO2 conversion process, particularly for the outdoor mass cultivation.

We evaluated the clinical characteristics, cytokine/chemokine concentrations, viral shedding and antibody kinetics in 30 patients with Middle East respiratory syndrome (MERS), including 6 non-survivors admitted to 3 MERS-designated hospitals. Old age, low albumin, altered mentality and high pneumonia severity index score at admission were risk factors for mortality. In addition, severe signs of inflammation at initial presentation (at hospital days 1-4), such as high inducible protein-10 (p=0.0013), monocyte chemoattractant protein-1 (p=0.0007) and interleukin 6 (p=0.0007) concentrations, and poor viral control (high viral load at hospital days 5–10, p

The purpose of the present double-blind randomized placebo-controlled clinical study was to investigate the effects of Passionflower on polysomnographic sleep parameters in subjects with insomnia disorder. A total number 110 adult participants (mean age = 40.47 ± 11.68, Female = 53.6%) met the inclusion criteria of insomnia disorder according to the DSM-5 (Diagnostic and Statistical Manual of Mental Disorders). After randomization, patients received either the Passionflower extract or the placebo for 2 weeks. Patients underwent an overnight polysomnography and completed sleep diaries, Insomnia Severity Index, and Pittsburgh Sleep Quality Index. Within group comparisons were analyzed with paired t-tests or Wilcoxon's signed rank tests, and between-group comparisons were analyzed with independent t-tests or Mann-Whitney U Tests, as appropriate. Total sleep time (TST) was significantly increased in the Passionflower group compared with placebo (Passionflower vs placebo, 23.05 ± 54.26 vs -0.16 ± 53.12; P = 0.049). Sleep efficiency and wake after sleep onset (WASO) significantly improved after 2 weeks in the Passionflower group but there was no difference compared with the placebo group. The current study demonstrated the positive effects of Passionflower on objective sleep parameters including TST on polysomnography in adults with insomnia disorder. Further study is needed to investigate the clinical efficacy of Passionflower on insomnia.

Regulatory T cells (Treg) are enriched in the tumor microenvironment (TME) and suppress antitumor immunity; however, the molecular mechanism underlying the accumulation of Tregs in the TME is poorly understood. In various tumor models, tumor-infiltrating Tregs were highly enriched in the TME and had significantly higher expression of immune checkpoint molecules. To characterize tumor-infiltrating Tregs, we performed bulk RNA sequencing (RNA-seq) and found that proliferation-related genes, immune suppression–related genes, and cytokine/chemokine receptor genes were upregulated in tumor-infiltrating Tregs compared with tumor-infiltrating CD4+Foxp3− conventional T cells or splenic Tregs from the same tumor-bearing mice. Single-cell RNA-seq and T-cell receptor sequencing also revealed active proliferation of tumor infiltrating Tregs by clonal expansion. One of these genes, ST2, an IL33 receptor, was identified as a potential factor driving Treg accumulation in the TME. Indeed, IL33-directed ST2 signaling induced the preferential proliferation of tumor-infiltrating Tregs and enhanced tumor progression, whereas genetic deletion of ST2 in Tregs limited their TME accumulation and delayed tumor growth. These data demonstrated the IL33/ST2 axis in Tregs as one of the critical pathways for the preferential accumulation of Tregs in the TME and suggests that the IL33/ST2 axis may be a potential therapeutic target for cancer immunotherapy.

The type II cohesin domain and type II dockerin of bacterial cellulosome were cloned from Clostridium thermocellum and expressed with the fusion of tobacco mosaic virus coat protein (TMVcp) and enhanced green fluorescent protein (EGFP), respectively, in Escherichia coli. The TMVcp-cohesin fusion protein was assembled to the stable and rod-shaped nanostructure (TMVcp-Coh rod) under a particular buffer condition, where many active cohesin proteins are biologically and densely displayed around the 3-dimensional surface of TMVcp-Coh rod. Using EGFP-dockerin as a fluorescent reporter, we confirmed that the Ca(2+)-dependent binding and dissociation between native cohesin and dockerin were reproduced with the two recombinant fusion proteins, TMVcp-cohesin and EGFP-dockerin. The multi-cyclic binding-dissociation operation of TMVcp-Coh rod and EGFP-dockerin was successfully performed with maintaining the reversible cohesin-dockerin interaction in every cycle. EGFP that was fused to dockerin as a proof-of-concept here can be switched to other functional proteins/peptides that need to be used in multi-cyclic operation.

Although protein nanoparticles (PNPs) (e.g., viral capsids) capable of delivering a broad range of drug agents have shown distinctive advantages over synthetic nanomaterials, PNPs have an intrinsic drawback that hampers their clinical application, that is, potential immunogenicity. Here, a novel method for resolving the immunogenicity problem of PNPs, which is based on the genetic presentation of albumin‐binding peptides (ABPs) on the surface of PNP, is reported. ABPs are inserted into the surface of a viral capsid (hepatitis B virus capsid/HBVC) while preserving the native self‐assembly function of HBVC. The ABPs effectively gather human serum albumins around HBVC and significantly reduce both inflammatory response and immunoglobulin titer in live mice compared to ABP‐free HBVC. Furthermore, ABP‐conjugated HBVCs remain within tumors for a longer period than HBVCs conjugated to tumor cell receptor‐bindingpeptides, indicating that the ABPs are also capable of enhancing tumor‐targeting performance. Although applied to HBVC for proof of concept, this novel approach may provide a general platform for resolving immunogenicity and cancer‐targeting problems of PNPs, which enables the development of a variety of PNP‐based drug delivery carriers with high safety and efficacy.

The hepatitis B virus (HBV) capsid-based recombinant particles, which display both major hydrophilic region of HBV surface antigen (HBV–MHR) and B domain of Staphylococcal protein A (SPAB), were produced using Escherichia coli as expression host. SPAB was used as an adjuvant to elicit the immune response to HBV–MHR, and its adjuvant effect in the immunized mice was estimated with varying the position and amount of SPAB on the HBV capsid particles. Compared to the emulsified aluminum gel (alum gel) that is a currently commercialized vaccine adjuvant, SPAB caused the significantly higher level of anti-HBV immunoglobulin G (IgG) titer and seroconversion rate, and notably SPAB at the most surface-exposed position on the recombinant particle led to the highest immune response. Moreover, SPAB caused much lower ratio of IgG1 to IgG2a compared to alum gel, indicating that helper T-cell 1-mediated immune response (responsible for cytotoxic T-cell stimulation) is relatively more stimulated by SPAB, unlike alum gel that mainly stimulates helper T-cell 2-mediated immune response (responsible for B-cell stimulation). Although HBV–MHR and HBV capsid particle were used as proof-of-concept in this study, SPAB can be used as a highly effective adjuvant with other disease-specific antigens on the surface of other virus-like particles to produce various recombinant vaccines with high potency. Biotechnol. Bioeng. 2015;9999: 1–7. © 2015 Wiley Periodicals, Inc.

Since the use of solubility enhancer proteins is one of the effective methods to produce active recombinant proteins within Escherichia coli, the development of a novel fusion expression partner that can be applied to various aggregation-prone proteins is of crucial importance. In our previous work, two-dimensional electrophoresis (2-DE) was employed to systematically analyze the E. coli BL21 (DE3) proteome profile in response to heat treatment, and KDPG aldolase (EDA) was identified as a heat-responsive and aggregation-resistant protein. When used as fusion expression partner, EDA significantly increased the solubility of seven aggregation-prone heterologous proteins in the E. coli cytoplasm. The efficacy of EDA as a fusion expression partner was evaluated through the analysis of bioactivity or secondary structure of several target proteins: EDA-fusion expression resulted in the synthesis of bioactive human ferritin light chain and bacterial arginine deiminase and the formation of correct secondary structure of human granulocyte colony stimulation factor.

Mesothelin (MSLN) is an antigen overexpressed in several malignancies, including mesothelioma and ovarian and pancreatic adenocarcinoma. It has been studied as a marker for diagnosis and a target for immunotherapy. Here, we adopted a bispecific antibody format for recruiting cytotoxic T cells to kill tumor cells. MG1122 is a novel, whole IgG-like bispecific antibody which recognizes CD3 on T cells and MSLN on tumor cells. MG1122 induced effective killing of MSLN-expressing tumor cells. This response was associated with robust activation of T cells as shown by nuclear factor of activated T cells (NFAT) activation, CD25 and CD69 upregulation and increased cytokine release. In addition, using a live cell analysis system, we found that levels of activated caspase 3/7 were amplified in the tumor cells by MG1122 treatment. Although MSLN is a surface antigen, it also exists as a secreted isoform, which can lead to tumor evasion against MSLN targeting antibodies. However, despite the high concentration of soluble MSLN, MG1122 still effectively bound and showed strong killing effects against MSLN-expressing tumor cells. Taken together, our MSLN/CD3 bispecific antibody, MG1122 offers a promising opportunity to redirect T cells to kill MSLN-expressing cancer cells. Citation Format: Yun-Jung Lee, Okjae Lim, Munkyung Kim, Jeewon Lee, Kisu Kim, Junhong Jung, SuA Lee, Sung Keun Kim, Haenaem Kwon, Yangmi Lim, Yong Yea Park, Jonghwa Won. MG1122, a whole IgG-like bispecific antibody targeting mesothelin and CD3, induces T cell-mediated killing of MSLN-expressing tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5768.

Previously, an age-predictive method based on DNA-methylation patterns in semen was developed, using three CpG sites (cg06304190 in the TTC7B gene, cg12837463, and cg06979108 in the NOX4 gene). Before considering the routine use of a new method in forensics, validation studies such as concordance and sensitivity tests are essential for obtaining expanded and more reliable forensic information. Here, we evaluated a previously described age-predictive method for semen for routine forensic use. Concordance testing showed a high correlation between the predicted and chronological age, with a mean absolute deviation from the chronological age of 4.8 years. Sensitivity testing suggested that age prediction with reliable accuracy and consistency was possible with >5 ng of bisulfite-converted DNA. We also confirmed the applicability of the age-predictive method in forensic casework, using forensic samples. Thus, the proposed method could serve as a very valuable forensics tool for accurate age prediction with semen samples.

Current immunoassays are in general performed through time-consuming multi-step procedures that depend on the use of premade signal-producing reporters and often cause assay inaccuracy. Here we report an advanced immunoassay technology that resolves the delayed, complex, and inaccurate assay problems of conventional immunoassays. We have developed an accurate, rapid, simple, and label-free one-step-immunoassay based on the self-enhancement of sensitive immunoassay signals in an assay solution. The nano-scale protein particles (hepatitis B virus capsid and human ferritin heavy chain particles) were genetically engineered to present many well-oriented antibody (or antigen) probes and multi-copies of poly-histidine peptides on their surface, resulting in the construction of 3-dimensional (3D) bioprobes that chemisorb gold ions via coordination bonding and sensitively detect both antigen and antibody analytes. Systematic numerical and experimental analyses show that the signal self-enhancement happens through two coupled reactions under reducing conditions: (1) 3D bioprobe-based sensitive immuno-detection of analytes and (2) coordinated assembly of free and chemisorbed gold nanoparticles around the 3D bioprobe-analyte-associated complexes, which is followed by the quick generation of apparent optical signals. This advanced one-step-immunoassay was successfully applied to diagnostic assays requiring high accuracy and/or speed, i.e. diagnosis of acute myocardial infarction and hepatitis C through detecting a cardiac protein (troponin I) and anti-hepatitis C virus antibodies in patient sera, indicating that it is applicable to the accurate and rapid detection of both antigen and antibody markers of a wide range of diseases.

DNA methylation has important biological roles, such as gene expression regulation, as well as practical applications in forensics, such as in body fluid identification and age estimation. DNA methylation often occurs in the CpG site, and methylation within the CpG islands affects various cellular functions and is related to tissue-specific identification. Several programs have been developed to identify CpG islands; however, the size, location, and number of predicted CpG islands are not identical due to different search algorithms. In addition, they only provide structural information for predicted CpG islands without experimental information, such as primer design. We developed an analysis pipeline package, CpGPNP, to integrate CpG island prediction and primer design. CpGPNP predicts CpG islands more accurately and sensitively than other programs, and designs primers easily based on the predicted CpG island locations. The primer design function included standard, bisulfite, and methylation-specific PCR to identify the methylation of particular CpG sites. In this study, we performed CpG island prediction on all chromosomes and compared CpG island search performance of CpGPNP with other CpG island prediction programs. In addition, we compared the position of primers designed for a specific region within the predicted CpG island using other bisulfite PCR primer programs. The primers designed by CpGPNP were used to experimentally verify the amplification of the target region of markers for body fluid identification and age estimation. CpGPNP is freely available at http://forensicdna.kr/cpgpnp/.

"Proteinticle" is a nano-scale protein particle that is self-assembled inside cells with constant 3D structure and surface topology. The binding of IgG to the B domain of Staphylococcal protein A (SPA(B)) molecules that are genetically inserted on the surface of proteinticle enables the variable domains of bound IgG to be well oriented to effectively capture antigens, accordingly forming a highly sensitive 3D IgG probe. The five different proteinticles that originate from humans, bacteria, and virus and totally differ in size, shape, and surface structure were used for the surface display of SPA(B). The dissociation constant (K(D)) in the binding of IgG to SPA(B) on the proteinticle surface was estimated based on the Langmuir adsorption isotherm model: K(D) was 1-3 orders-of-magnitude lower compared to the previously reported K(D) in the binding of IgG to Staphylococcal protein A. The surface density and distribution of SPA(B) and especially the existence of hot (or highly congested) spots of SPA(B), which depend on the surface structure and the number of subunits as well as size and shape of proteinticle, is of crucial importance for the effective binding of IgG to SPA(B) on proteinticles. Although the five different proteinticles were demonstrated as proof-of-concept here, SPA(B)-mediated immobilization of IgG on the other proteinticles would be very useful for the fabrication of sensitive 3D immunoassay platforms.

When used as an N-terminal fusion expression partner, the Escherichia coli stress-responsive protein, CysQ dramatically increased the cytoplasmic solubility of various aggregation-prone heterologous proteins: Pseudomonas putida cutinase (CUT), human granulocyte colony-stimulating factor (hG-CSF), human ferritin light chain (hFTN-L), arginine deiminase (ADI), human interleukin-2 (IL2), human activation induced cytidine deaminase (AID), and deletion mutant of human glutamate decarboxylase (GAD448-585). As compared with well-known fusion tags such as glutathione-S-transferase (GST) and maltose-binding protein (MBP), the performance of CysQ as solubility enhancer was evidently better than GST and was similar to or better than MBP for the seven heterologous proteins above. This is likely due to the intrinsic ability of CysQ to form its native conformation, probably promoting the binding of molecular chaperones during the folding of CysQ-fusion protein. When used as a substrate, p-nitrophenyl butyrate (PNB) was successfully hydrolyzed to p-nitrophenol by CysQ-CUT fusion mutant. Even after CysQ was removed, the solubility of hFTN-L and hG-CSF, the secondary structure of hG-CSF, and self-assembly activity of hFTN-L were successfully maintained. Conclusively, it seems that CysQ is a highly effective solubility enhancer and fusion expression partner for the production of a variety of bio-active recombinant proteins.

We found Escherichia coli proteins, elongation factor Ts (Tsf), and malate dehydrogenase (Mdh) that can exist in the form of native and soluble proteins even under stress situation such as heat shock and protein denaturing condition. To examine their property as solubility enhancers, aggregation-prone Mycoplasma arginine deiminase (mADI), which has been suggested as anti-cancer agent, was fused to the C-terminal of each of them and cloned into pET28a to be expressed in the E. coli cytoplasm. When mADI was fused to fusion partners (Mdh, Tsf), a significant portion of the recombinant mADI was expressed in a soluble fraction (>90%) whereas the directly expressed mADI was aggregated to the inclusion body. In addition, recombinant mADI released from the fusion tag retained its soluble form and presented its specific enzymatic activity of converting l-arginine into citrulline (>10 U/mg). These results show that Tsf and Mdh could serve as effective solubility enhancers for aggregation-prone proteins (e.g. mADI in this case) when used as fusion expression partners in bacterial expression systems.

The conversion of methane to methanol is important to economic utilization of natural/shale gas. Methanol is a valuable liquid fuel and raw material for various synthetic hydrocarbon products. Its industrial production is currently based on a two-step process that is energy-intensive and environmentally unfriendly, requiring high pressure and temperature. The biological oxidation of methane to methanol, based on methane monooxygenase activity of methanotrophic bacteria, is desirable because the oxidation is highly selective under mild conditions, but conversion rate and yield and stability of catalytic activity should be improved up to an industrially viable level. Since methanotrophic bacteria produce methanol as only a precursor of formaldehyde that is then used to synthesize various essential metabolites, the direct use of bacteria seems unsuitable for selective production of a large amount of methanol. There are two types of methane monooxygenase: soluble (sMMO) and particulate (pMMO) enzyme. sMMO consisting of three components (reductase, hydroxylase, and regulatory protein) features an (αβγ)2 dimer architecture with a di-iron active site in hydroxlase. pMMO, a trimer (pmoA, pmoB, and pmoC) in an α 3 β 3 γ 3 polypeptide arrangement is a copper enzyme with a di-copper active site located in the soluble domain of pmoB subunit. Since the membrane transports electrons well and delivers effectively methane with increased solubility in the lipid bilayer, pMMO seems more rationally designed enzyme in nature than sMMO. The engineering/evolution/modification of MMO enzymes using various biological and chemical techniques could lead to an optimal way to reach the ultimate goal of technically and economically feasible and environmentally friendly oxidation of methane. For this, multidisciplinary efforts from chemical engineering, protein engineering, and bioprocess research sectors should be systematically combined.

Acute hepatitis A caused by hepatitis A virus (HAV) infection is accompanied by severe liver injury in adult patients, and the liver injury is associated with the production of chemokines. Herein, we investigated the mechanism of how HAV infection induces the production of CXCR3 and CCR5 chemokines, such as CXCL10, CCL4 and CCL5. The production of CXCL10, CCL4 and CCL5 was markedly increased by HAV (HM-175/18f) infection in the culture of primary human hepatocytes and HepG2 cells. In particular, CXCL10 was produced in HAV-infected cells, not in neighboring uninfected cells. Moreover, these chemokines were significantly increased in the sera of acute hepatitis A patients. The production of IFN-λs was also robustly induced by HAV infection, and the blocking of secreted IFN-λs partially abrogated the production of CCL4 and CCL5 in HAV-infected cells. However, CXCL10 production was not decreased by the blocking of IFN-λs. Instead, CXCL10 production was reduced by silencing the expression of RIG-I-like receptor (RLR) signal molecules, such as mitochondrial antiviral signaling protein and interferon regulatory factor 3, in HAV-infected cells. In conclusion, HAV infection strongly induces the production of helper 1 T cell-associated chemokines, particularly CXCL10 via RLR signaling, even without secreted IFNs.

Human ferritin heavy-chain nanoparticle (hFTH) is genetically engineered to present tumor receptor-binding peptides (affibody and/or RGD-derived cyclic peptides, named 4CRGD here) on its surface. The affibody and 4CRGD specifically and strongly binds to human epidermal growth factor receptor I (EGFR) and human integrin αvβ3, respectively, which are overexpressed on various tumor cells. Through in vitro culture of EGFR-overexpressing adenocarcinoma (MDA-MB-468) and integrin-overexpressing glioblastoma cells (U87MG), it is clarified that specific interactions between receptors on tumor cells and receptor-binding peptides on engineered hFTH is critical in active tumor cell targeting. After labeling with the near-infrared fluorescence dye (Cy5.5) and intravenouse injection into MDA-MB-468 or U87MG tumor-bearing mice, the recombinant hFTHs presenting either peptide or both of affibody and 4CRGD are successfully delivered to and retained in the tumor for a prolonged period of time. In particular, the recombinant hFTH presenting both affibody and 4CRGD notably enhances in vivo detection of U87MG tumors that express heterogeneous receptors, integrin and EGFR, compared to the other recombinant hFTHs presenting either affibody or 4CRGD only. Like affibody and 4CRGD used in this study, other multiple tumor receptor-binding peptides can be also genetically introduced to the hFTH surface for actively targeting of in vivo tumors with heterogenous receptors.

Efficient delivery of tumor-specific antigens (TSAs) to lymph nodes (LNs) is essential to eliciting robust immune response for cancer immunotherapy but still remains unsolved. Herein, we evaluated the direct LN-targeting performance of four different protein nanoparticles with different size, shape, and origin [Escherichia coli DNA binding protein (DPS), Thermoplasma acidophilum proteasome (PTS), hepatitis B virus capsid (HBVC), and human ferritin heavy chain (hFTN)] in live mice, using an optical fluorescence imaging system. Based on the imaging results, hFTN that shows rapid LN targeting and prolonged retention in LNs was chosen as a carrier of the model TSA [red fluorescence protein (RFP)], and the flexible surface architecture of hFTN was engineered to densely present RFPs on the hFTN surface through genetic modification of subunit protein of hFTN. The RFP-modified hFTN rapidly targeted LNs, sufficiently exposed RFPs to LN immune cells during prolonged period of retention in LNs, induced strong RFP-specific cytotoxic CD8+ T cell response, and notably inhibited RFP-expressing melanoma tumor growth in live mice. This suggests that the strategy using protein nanoparticles as both TSA-carrying scaffold and anti-cancer vaccine holds promise for clinically effective immunotherapy of cancer.

Down syndrome is the most common genetic disorder and is characterized by three copies of chromosome 21. Regulator of calcineurin 1 (RCAN1) is located close to the Down syndrome critical region (distal part of chromosome 21), and its product functions as an endogenous inhibitor of calcineurin signaling. RCAN1 protein stability is regulated by several inflammatory signaling factors, though the underlying mechanisms remain incompletely understood. Here, we report that RCAN1 interacts with the inflammation-linked transcription factor, signal transducer and activator of transcription 2 (STAT2) in mammalian cells. STAT2 overexpression decreased levels of RCAN1 protein. Decreases in RCAN1 were blocked by a proteasome inhibitor, indicating that STAT2 regulates RCAN1 degradation via the ubiquitin-proteasome system. Co-immunoprecipitation/immunoblot analyses showed that STAT2 enhanced RCAN1 ubiquitination through the ubiquitin E3 ligase FBW7. This pathway appeared to be physiologically relevant, as treatment of cells with interferon-α reduced RCAN1 levels through the activation of STAT2 and FBW7. Together, these results suggest that STAT2 influences diverse cellular processes linked to RCAN1 by negatively affecting RCAN1 protein stability.

As a fusion partner to express aggregation-prone heterologous proteins, we investigated the efficacy of Escherichia coli phosphoglycerate kinase (ePGK) that consists of two functional domains (N- and C-domain) and reportedly has a high structural stability. When the full-length ePGK (F-ePGK) was used as a fusion partner, the solubility of the heterologous proteins increased, but some of them still had a large fraction of insoluble aggregates. Surprisingly, the fusion expression using the N-domain of ePGK (N-ePGK) made the insoluble fraction significantly reduce to less than 10% for all the heterologous fusion proteins tested. Also, we evaluated the efficacy of N-ePGK in making the target proteins be expressed with their own native function or structure. It was found that of human ferritin light chain, bacterial arginine deiminase, human granulocyte colony stimulating factor were synthesized evidently with the self-assembly function, L-arginine-degrading activity, and the correct secondary structure, respectively, through the fusion expression using N-ePGK. These results indicate that N-ePGK is a highly potent fusion partner that can be widely used for the synthesis of a variety of heterologous proteins in E. coli. Biotechnol. Bioeng. 2012; 109:325–335. © 2011 Wiley Periodicals, Inc.

Through the proteome analysis of Escherichia coli BL21(DE3), we previously identified the stress-responsive protein, arsenate reductase (ArsC), that showed a high cytoplasmic solubility and a folding capacity even in the presence of stress-inducing reagents. In this study, we used ArsC as an N-terminal fusion partner to synthesize nine aggregation-prone proteins as water-soluble forms. As a result, solubility of the aggregation-prone proteins increased dramatically by the fusion of ArsC, due presumably to its tendency to facilitate the folding of target proteins. Also, we evaluated and confirmed the efficacy of ArsC-fusion expression in making the fusion-expressed target proteins have their own native function or structure. That is, the self-assembly function of human ferritin light chain, l-arginine-degrading function of arginine deiminase, and the correct secondary structure of human granulocyte colony stimulating factor were clearly observed through transmission electron microscope analysis, colorimetric enzyme activity assay, and circular dichroism, respectively. It is strongly suggested that ArsC can be in general an efficient fusion expression partner for the production of soluble and active heterologous proteins in E. coli.

Existing methods to enrich target regions of genomic DNA based on PCR, hybridization capture, or molecular inversion probes have various drawbacks, including long experiment times and low throughput and/or enrichment quality. We developed CRISPR-Cap, a simple and scalable CRISPR-based method to enrich target regions of dsDNA, requiring only two short experimental procedures that can be completed within two hours. We used CRISPR-Cap to enrich 10 target genes 355.7-fold on average from Escherichia coli genomic DNA with a maximum on-target ratio of 81% and high enrichment uniformity. We also used CRISPR-Cap to measure gene copy numbers and detect rare alleles with frequencies as low as 1%. Finally, we enriched coding sequence regions of 20 genes from the human genome. We envision that CRISPR-Cap can be used as an alternative to other widely used target-enrichment methods, which will broaden the scope of CRISPR applications to the field of target enrichment field.

－ Recent in vitro and in vivo animal studies have reported that statin, a cholesterol-lowering drug, stimulate osteogenic differentiation. In the present study, we investigated the effect of simvastatin on osteogenic and adipogenic differentiation in primarily cultured human adipose-derived stem cells (hADSCs). The simvastatin treatment significantly increased the positive cell numbers in alkaline phosphatase and von Kossa staining, and enhanced the expression levels of bone morphogenic protein (BMP)-2, core binding factor alpha 1 (cbfa1), collgen type I and osteonectin mRNAs. Lastly, hADSCs were cultured in the adipo-genic media with or without simvastatin to examine the effect of simvastatin on adipogenic differentiation. In the RT-PCR analysis, there were notable decreases in mRNA expression of aP1, C/EBP-α and PPAR-γ in hADSCs cultivated in simvastatin-added medium, compared to those in simvastatin-free medium. It suggests that the adipogenic differentiation was significantly inhibited by simvastatin treatment. These observations indicate that simvastatin induces osteogenic differentiation and suppresses adipogenic differentiation in hADSCs.Keywords: Simvastatin, Human adipose-derived stem cells, Osteogenic differentiation, Adipogenic differ-entiation

We report for the first time that the C-terminal region of hG-CSF suffers from proteolytic degradation when human granulocyte colony-stimulating factor (hG-CSF) is directly expressed in Escherichia coli BL21(DE3). It is believed that the rapid proteolysis occurs at the C-terminus of hG-CSF that is very easily exposed to E. coli protease(s) during a short period following protein synthesis and prior to completion of the formation of the inclusion body. The recombinant hG-CSF that is expressed with an N-terminal fusion partner is effectively protected from the proteolysis. It seems that since the N-terminus of hG-CSF is located very close to the C-terminus, the presence of the N-terminal fusion partner masks the C-terminal region of hG-CSF and protects it from proteolytic degradation by E. coli protease(s). Furthermore, the solubility of hG-CSF markedly increased in E. coli cytoplasm when a stress-responsive and aggregation-resistant protein, i.e. aspartate carbamoyl-transferase catalytic chain (PyrB) was used as a novel N-terminal fusion partner proteins.

We previously reported that under the stress condition caused by the addition of 2-hydroxyethyl disulfide, a thiol-specific oxidant, to growing cultures of Escherichia coli BL21(DE3), a population of stress-responsive proteins [peptidyl-prolyl cis–trans isomerase B (PpiB), bacterioferritin (Bfr), putative HTH-type transcriptional regulator yjdC (YjdC), dihydrofolate reductase (FolA), chemotaxis protein cheZ (CheZ), and glutathione synthetase (GshB)] were significantly upregulated when compared with the nonstress condition. When those stress-responsive proteins were used as fusion partners for the expression of human granulocyte colony-stimulating factor (hG-CSF), the solubility of hG-CSF was dramatically enhanced in E. coli cytoplasm, whereas almost all of the directly expressed hG-CSF were aggregated to inclusion bodies. In addition, the spectra of circular dichroism measured with the purified hG-CSF were identical to that of standard hG-CSF, implying that the synthesized hG-CSF has native conformation. These results indicate that the bacterial stress-responsive proteins could be potent fusion expression partners for aggregation-prone heterologous proteins in E. coli cytoplasm.

Early detection of the protein marker troponin I in patients with a higher risk of acute myocardial infarction can reduce the risk of death from heart attacks. Most troponin assays are currently based on the conventional enzyme linked immunosorbent assay and have detection limits in the nano- and picomolar range. Here, we show that by combining viral nanoparticles, which are engineered to have dual affinity for troponin antibodies and nickel, with three-dimensional nanostructures including nickel nanohairs, we can detect troponin levels in human serum samples that are six to seven orders of magnitude lower than those detectable using conventional enzyme linked immunosorbent assays. The viral nanoparticle helps to orient the antibodies for maximum capture of the troponin markers. High densities of antibodies on the surfaces of the nanoparticles and nanohairs lead to greater binding of the troponin markers, which significantly enhances detection sensitivities. The nickel nanohairs are re-useable and can reproducibly differentiate healthy serum from unhealthy ones. We expect other viral nanoparticles to form similar highly sensitive diagnostic assays for a variety of other protein markers.

Bacillus sp. SB-007 was isolated from pea leaves harvested from the southwestern parts of South Korea through screening on a minimal medium containing 0.2% purified cutin for its ability to induce the cutinase production. However, no cutinase was produced when it was grown in a minimal medium containing 0.2% glucose. A proteomic approach was applied to separate and characterize these differentially secreted proteins. The expression level of 83 extracellular proteins of the cutinase-producing Bacillus sp. strain SB-007 incubated in a cutinase-induced medium increased significantly as compared with that cultured in a non cutinase-induced medium containing glucose. The extracellular proteome of Bacillus sp. SB-007 includes proteins from different functional classes, such as enzymes for the degradation of various macromolecules, proteins involved in energy metabolism, sporulation, transport/binding proteins and lipoproteins, stress inducible proteins, several cellular molecule biosynthetic pathways and catabolism, and some proteins with an as yet unknown function. In addition, the two protein spots showed little similarities with the known lipolytic enzymes in the database. These secreted proteome analysis results are expected to be useful in improving the Bacillus strains for the production of industrial cutinases.

The Escherichia coli proteome response to the stressor GdnHCl was analyzed through 2-dimensional gel electrophoresis (2-DE). We identified PotD (spermidine/putrescine-binding periplasmic protein) and Crr [glucose-specific phosphotransferase (PTS) enzyme IIA component] as a stress-responsive protein. Even under a stress situation where the total number of soluble proteins decreased by about 10%, 3.5- and 2.2-fold increase was observed in the synthesis of PotD and Crr, respectively. As fusion partners, PotD and Crr dramatically increased the solubility of many aggregation-prone heterologous proteins [e.g. human minipro-insulin (mp-INS), human epidermal growth factor (EGF), human prepro-ghrelin (ppGRN), human interleukin-2(hIL-2), human activation induced cytidine deaminase (AID), human glutamate decarboxylase (GAD(448-585)), Pseudomonas putida cutinase (CUT), human ferritin light chain (hFTN-L), human granulocyte colony-stimulating factor (G-CSF), and cold autoinflammatory syndrome1 protein (NALP3) Nacht domain (NACHT)] in the E. coli cytoplasm. Presumably PotD and Crr were very effective in shielding interactive surfaces of heterologous proteins associated with non-specific protein-protein interactions leading to the formation of inclusion bodies most likely due to intrinsic high folding efficiency, chaperone-like activity, or a combination of both factors. Both the stress-induced proteins were well suited for the production of a biologically active fusion mutant of P. putida cutinase that can be expected to be of biotechnological and commercial interest.

E. coli proteome response to the stressor 2-HEDS was analyzed through two-dimensional gel electrophoresis (2-DE), and we identified DNA-directed RNA polymerase α-subunit (RpoA) as stress-responsive protein. Even under stress situation where the total number of soluble proteins decreased by 9.8%, the synthesis level of RpoA was increased 1.5-fold. As a fusion expression partner as well as solubility enhancer, RpoA facilitated the folding and increased significantly the solubility of many aggregation-prone heterologous proteins (human minipro-insulin, human epidermal growth factor, human prepro-ghrelin, human interleukin-2, human activation induced cytidine deaminase, human glutamate decarboxylase, Pseudomonas putida cutinase, human ferritin light chain, human granulocyte colony-stimulating factor, and cold inflammatory syndrome1 protein Nacht domain) in E. coli cytoplasm. Due probably to intrinsic high folding efficiency and/or chaperone-like activity, RpoA was very effective in shielding interactive surfaces of heterologous proteins that are associated with non-specific protein–protein interaction leading to the formation of inclusion bodies. RpoA was also well suited for the production of biologically active fusion mutant of Pseudomonas putida cutinase that is of much biotechnological and commercial interest.

Through two-dimensional electrophoresis, Escherichia coli proteome response to a protein denaturant, guanidine hydrochloride, was analyzed and elongation factor Ts (Tsf) detected as a stress-induced protein. Many host proteins aggregated, or their synthesis levels decreased significantly under conditions of protein denaturation as 34 out of 699 soluble proteins knocked out and 63 proteins decreased by over 2.5-fold. Interestingly, the expression level of Tsf increased 1.61-fold compared with a nonstress condition. Contrary to direct expression, various heterologous proteins were solubly expressed in E. coli when subjected to N-terminus fusions of Tsf. Owing most likely to an intrinsic high folding efficiency, Tsf seemed to play critical roles in sequestering interactive surfaces of heterologous proteins from nonspecific protein-protein interactions leading to formation of inclusion bodies. It has been also demonstrated that Tsf is effective in aiding the production of a biologically active bacterial cutinase, which could be of interest to biotechnology and commercial applications.

An ustilaginomycetous anamorphic yeast, isolated from orange leaves on Jeju island in South Korea, represents a novel Pseudozyma species according to morphologic and physiologic findings and molecular taxonomic analysis using the D1/D2 domains of the large subunit (26S) rRNA gene and the internally transcribed spacer (ITS) 1+2 regions. The name Pseudozyma jejuensis sp. nov. is proposed for this novel species, with OL71(T) (=KCTC 17482(T)=CBS 10454(T)) as type strain. In the present study, we have also demonstrated that Pseudozyma jejuensis OL71 is capable of producing cutinase and degrading polycaprolactone. These results suggest that Pseudozyma jejuensis or its cutinase may be useful for the biological degradation of plastic waste.

Recently, we reported on the dual function of human ferritin heavy chain (hFTN-H) used for the fusion expression and solubility enhancement of various heterologous proteins: (1) high-affinity interaction with HSP70 chaperone DnaK and (2) formation of self-assembled supramolecules with limited and constant sizes. Especially the latter, the self-assembly function of hFTN-H is highly useful in avoiding the undesirable formation of insoluble macroaggregates of heterologous proteins in bacterial cytoplasm. In this study, using enhanced green fluorescent protein (eGFP) and several deletion mutants of Mycoplasma arginine deiminase (ADI(132-410)) as reporter proteins, we confirmed through TEM image analysis that the recombinant fusion proteins (hFTN-H::eGFP and hFTN-H::ADI(132-410)) formed intracellular spherical particles with nanoscale diameter ( approximately 10 nm), i.e., noncovalently cross-linked supramolecules. Surprisingly, the supramolecular eGFP and ADI showed much enhanced stability in bioactivity. That is, the activity level was much more stably maintained for the prolonged period of time even at high temperature, at high concentration of Gdn-HCl, and in wide range of pH. The stability enhancement by supramolecular self-assembly may make it possible to utilize the protein supramolecules as novel means for drug delivery, enzymatic material conversion (biotransformation), protein chip/sensor, etc. where the maintenance of protein/enzyme stability is strictly required.

Interpreting epistatic interactions is crucial for understanding evolutionary dynamics of complex genetic systems and unveiling structure and function of genetic pathways. Although high resolution mapping of en masse variant libraries renders molecular biologists to address genotype-phenotype relationships, long-read sequencing technology remains indispensable to assess functional relationship between mutations that lie far apart. Here, we introduce JigsawSeq for multiplexed sequence identification of pooled gene variant libraries by combining a codon-based molecular barcoding strategy and de novo assembly of short-read data. We first validate JigsawSeq on small sub-pools and observed high precision and recall at various experimental settings. With extensive simulations, we then apply JigsawSeq to large-scale gene variant libraries to show that our method can be reliably scaled using next-generation sequencing. JigsawSeq may serve as a rapid screening tool for functional genomics and offer the opportunity to explore evolutionary trajectories of protein variants., This paper described a new and efficient method for de novo assembly of multiple DNA sequence information from mutagenized clone libraries. Using codon-barcoded libraries and calling the method JigsawSeq, the authors overcome limitations of short-read sequencing assembly from next-generation sequencing.

Genome engineering can be used to produce bacterial strains with a wide range of desired phenotypes. However, the incorporation of gene-sized DNA fragments is often challenging due to the intricacy of the procedure, off-target effects and low insertion efficiency. Here we report a genome engineering method enabling the continuous incorporation of gene-sized double-stranded DNAs (dsDNAs) into the Escherichia coli genome. DNA substrates are inserted without introducing additional marker genes, by synchronously turning an endogenous counter-selectable marker gene ON and OFF. To accomplish this, we utilized λ Red protein-mediated recombination to insert dsDNAs within the promoter region of a counter-selectable marker gene, tolC. By repeatedly switching the marker gene ON and OFF, a number of desired gene-sized dsDNAs can be inserted consecutively. With this method, we successfully inserted approximately 13 kb gene clusters to generate engineered E. coli strains producing 1,4-butanediol (1,4-BDO).

The efficiency of two lipolytic enzymes (fungal cutinase and yeast esterase) in the degradation of dipentyl phthalate (DPeP) was investigated. The DPeP degradation rate of fungal cutinase was surprisingly high, i.e., almost 60% of the initial DPeP (500 mg/L) was decomposed within 2.5 hours, and nearly 40% of the degraded DPeP disappeared within the initial 15 minutes. With the yeast esterase, despite the same concentration, >87% of the DPeP remained even after 3 days of treatment. The final chemical composition after 3 days was significantly dependent on the enzyme used. During degradation with cutinase, most DPeP was converted into 1,3-isobenzofurandione (IBF) by diester hydrolysis. However, in the degradation by esterase, pentyl methyl phthalate, in addition to IBF, was produced in abundance. Toxicity monitoring using various recombinant bioluminescent bacteria showed that the degradation products from yeast esterase contained a toxic hazard, causing oxidative stress and damage to protein synthesis.

During fed-batch cultivation of Escherichia coli K-12, the proteomic response to a temperature downshift from 37 to 20 degrees C was quantitatively monitored and analyzed by using two-dimensional electrophoresis. When the temperature of exponentially growing E. coli K-12 culture was downshifted to 20 degrees C, the synthesis level of 57 intracellular proteins showed significant changes for a prolonged period of time, compared to the fed-batch culture controlled at 37 degrees C. Thus, these proteins are regarded as important stress proteins responsive to cold shock, which were analyzed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and identified using the E. coli SWISS-2DPAGE database. Most of the identified proteins were shown to be involved in energy metabolism, several cellular molecule biosynthetic pathways and catabolism, cell processes, flagellar biosynthesis and motility, and protein translation and folding. The systematic approach to the monitoring of proteomic responses and the detailed analysis results reported in this article would be useful in understanding the metabolic adaptation to lowered culture temperature and designing efficient fermentation strategies for the production of recombinant proteins and metabolites using E. coli strains.

Efficiencies of two lypolytic enzymes (fungal cutinase and yeast esterase) in malathion degradation were investigated. Surprisingly, degradation rate of malathion by fungal cutinase was very high, i.e. almost 60% of initial malathion (500 mg l(-1)) was decomposed within 0.5 h, and nearly 50% of the degraded malathion disappeared within initial 15 min. With the yeast esterase, despite the same concentration, more than 65% of malathion remained even after 2-day treatment. During enzymatic degradation of malathion, two malathion-derived compounds were detected, and time-course changes in composition were also monitored. In the degradation by both fungal cutinase and yeast esterase, two additional organic chemicals were produced from malathion: malathion monoacid (MMA) and malathion diacid (MDA) by ester hydrolysis. Final chemical composition after 2 d was significantly dependent on the enzyme used. Fungal cutinase produced MDA as a major degradation compound. However in the malathion degradation by yeast esterase, an isomer of MMA was produced in abundance in addition to MDA. Toxic effects of malathion and its final degradation products were investigated using various recombinant bioluminescent bacteria. As a result, the degradation products (including MMA) by esterase severely caused membrane damage and inhibition of protein synthesis in bacterial cells, while in the fungal cutinase processes, malathion was significantly degraded to non-toxic MDA after the extended period (2 days).