Cell-free protein synthesis (CFPS) systems based on crude cell extracts have been used in protein expression in vitro. With the researchers' endeavor for decades, the CFPS system has been developed as an important research tool in many frontiers of fundamental and applied biology because of its clear genetic background and simplicity to control the reaction. The yield of CFPS systems derived from prokaryote or eukaryote has increased to several grams per liter with constantly decreasing cost. Nowadays grams of protein could be prepared using a large-scale cell-free system. Recently, the advantages on the expression of complicated, toxic and membrane proteins have shown the great potential of the CFPS systems. The rapid progress of this technology made us to believe that it will take an important place in biopharmaceutical industries undoubtedly.
Bioengineering ; trends ; Cell-Free System ; Drug Industry ; trends ; Protein Biosynthesis

Cell-free synthetic biology system can perform biological transcription and translation process in vitro. Because of its advanced features, such as flexible openness, easy control, short expression time and high tolerance to cytotoxicity, this systemhas been successfully used to synthesize proteins that are difficult to express in cells. With the continuous development of cell-free biosensing technology and the lyophilization technology, its applications have widely expanded into many biomedical fields. This review discusses the current research progress of cell-free synthetic biology system in on-demand biopharmaceutical synthesis, portable diagnostics, and others. Further development of the system can lead to even more complicated synthesis of therapeutic proteins with post-translational modifications and evolution of different cell-free biosensors with high sensitivity. Cell-free synthetic biology as an emerging engineering strategy can be a better means applied to high-throughput screening of pharmaceutical proteins, detection of new pathogens, and other important health-care fields in the future.
Biosensing Techniques ; Cell-Free System ; Industry ; Synthetic Biology

Cell-free fetal DNA in maternal plasma of pregnant woman, originated from fetal and / or placental cells undergoing apoptosis, is mainly the short-sized DNA fragments of less than 313 base pairs in length for the sake of nuclear endonuclease selectively cleaving fetal DNA during the apoptosis process. The mean cell-free circulating fetal DNA in maternal plasma accounted for 3.4% and 6.2% of plasma total DNA during the early and the late gestation, respectively. Owing to its relative abundance, circulating fetal DNA in maternal plasma has now become the important DNA source for non-invasive prenatal molecular genetic diagnosis and it is widely used in fetal sex-determination, detection of fetal Rh (D) sequence in the plasma of the rhesus-negative woman, fetal aneuploidy detection, fetal STR genotyping and other clinical applications. Cell-free fetal DNA source, concentration, purity, size, distributions and postnatal clearance of fetal DNA in maternal plasma as well as the reported clinical applications are summarized and discussed in this paper. Based on the molecular characteristics of cell-free fetal DNA and the target gene, the using of appropriate molecular diagnosis strategy and experimental design as well as reducing the fragment size of PCR product and adjusting the PCR conditions to the optimum enable the improvement of non-invasive prenatal diagnosis accuracy.
Cell-Free System ; DNA ; blood ; metabolism ; Fetus ; metabolism ; Genetic Diseases, Inborn ; diagnosis ; genetics ; Humans ; Mothers

Recently, in situ protein microarrays have been developed for large scale analysis and high throughput studies of proteins. In situ protein microarrays produce proteins directly on the solid surface from pre-arrayed DNA or RNA. The advances in in situ protein microarrays are exemplified by the ease of cDNA cloning and cell free protein expression. These technologies can evaluate, validate and monitor protein in a cost effective manner and address the issue of a high quality protein supply to use in the array. Here we review the importance of recently employed methods: PISA (protein in situ array), DAPA (DNA array to protein array), NAPPA (nucleic acid programmable protein array) and TUSTER microarrays and the role of these methods in proteomics.
Cell-Free System ; DNA ; metabolism ; Oligonucleotide Array Sequence Analysis ; Protein Array Analysis ; Proteins ; metabolism ; RNA ; metabolism

The purpose of this study was to estimate the possibilities of an acellular matrix using a modified acellularization protocol, which circumvents immunological, microbiological, and physiological barriers. We treated porcine subclavian arteries with various reagents to construct acellular grafts. Afterwards, these grafts were interposed in a mongrel dogs' abdominal aorta. Six dogs underwent interposition with fresh porcine grafts (control group), and seven had interposed acellular grafts (acellular group). The control and acellular group dogs were sacrificed at 1, 3, 5 (n=2 in each group) and 12 months (n=1 in acellular group) after the operation. Histopathological examinations were then performed, to assess the degree to which re-endothelialization, inflammation, thrombus formation, and calcification occurred. The entire acellular group, but none of the control group, exhibited re-endothelialization. The degrees to which inflammation, thrombosis, and calcification occurred were found to be lower in the acellular group. We also discovered many smooth muscle cells in the medial layer of the xenograft that had been implanted in the dog sacrificed 12 months after the operation. These results suggest that the construction of xenografts using our modified acellularization protocol may offer acceptable outcomes as a vascular xenograft.
Transplantation, Heterologous/*methods ; Tissue Engineering/*methods ; Swine ; Subclavian Artery/*cytology/*transplantation ; Graft Survival/*physiology ; Dogs ; Cell-Free System/*transplantation ; Animals

The major bottle-neck in the way of constructing high density protein microarray is the availability and stability of proteins. The traditional methods of generating protein arrays require the in-vivo expression, purification and immobilization of hundreds or thousands of proteins. The cell-free protein array technology employs cell-free expression systems to produce proteins directly onto surface from co-distributed or pre-arrayed DNA or RNA, thus avoiding the laborious and often costly processes of protein preparation in the traditional approach. Here we provide an overview of recently developed novel technology in cell free based protein microarray and their applications in protein interaction analysis, in antibody specificity and vaccine screening, and in biomarker assay.
Cell-Free System ; DNA ; genetics ; Humans ; Protein Array Analysis ; methods ; Protein Biosynthesis ; Protein Interaction Mapping ; Proteins ; analysis ; genetics ; metabolism

Vasicinone, a quinazoline alkaloid from Adhatoda vasica Nees. is well known for its bronchodilator activity. However its antiproliferative activities is yet to be elucidated. Here-in we investigated the anti-proliferative effect of vasicinone and its underlying mechanism against A549 lung carcinoma cells. The A549 cells upon treatment with various doses of vasicinone (10, 30, 50, 70 µM) for 72 h showed significant decrease in cell viability. Vasicinone treatment also showed DNA fragmentation, LDH leakage, and disruption of mitochondrial potential, and lower wound healing ability in A549 cells. The Annexin V/PI staining showed disrupted plasma membrane integrity and permeability of PI in treated cells. Moreover vasicinone treatment also lead to down regulation of Bcl-2, Fas death receptor and up regulation of PARP, BAD and cytochrome c, suggesting the anti-proliferative nature of vasicinone which mediated apoptosis through both Fas death receptors as well as Bcl-2 regulated signaling. Furthermore, our preliminary studies with vasicinone treatment also showed to lower the ROS levels in A549 cells and have potential free radical scavenging (DPPH, Hydroxyl) activity and ferric reducing power in cell free systems. Thus combining all, vasicinone may be used to develop a new therapeutic agent against oxidative stress induced lung cancer.
Apoptosis ; Cell Membrane ; Cell Survival ; Cell-Free System ; Cytochromes c ; DNA Fragmentation ; Down-Regulation ; Justicia ; Lung Neoplasms ; Lung* ; Oxidative Stress ; Permeability ; Receptors, Death Domain ; Up-Regulation ; Wound Healing

Glutamate toxicity-mediated mitochondrial dysfunction and neuronal cell death are involved in the pathogenesis of several neurodegenerative diseases as well as acute brain ischemia/stroke. In this study, we investigated the neuroprotective mechanism of dieckol (DEK), one of the phlorotannins isolated from the marine brown alga Ecklonia cava, against glutamate toxicity. Primary cortical neurons (100 µM, 24 h) and HT22 neurons (5 mM, 12 h) were stimulated with glutamate to induce glutamate toxic condition. The results demonstrated that DEK treatment significantly increased cell viability in a dose-dependent manner (1–50 µM) and recovered morphological deterioration in glutamate-stimulated neurons. In addition, DEK strongly attenuated intracellular reactive oxygen species (ROS) levels, mitochondrial overload of Ca²⁺ and ROS, mitochondrial membrane potential (ΔΨ(m)) disruption, adenine triphosphate depletion. DEK showed free radical scavenging activity in the cell-free system. Furthermore, DEK enhanced protein expression of heme oxygenase-1 (HO-1), an important anti-oxidant enzyme, via the nuclear translocation of nuclear factor-like 2 (Nrf2). Taken together, we conclude that DEK exerts neuroprotective activities against glutamate toxicity through its direct free radical scavenging property and the Nrf-2/HO-1 pathway activation.
Adenine ; Brain ; Cell Death ; Cell Survival ; Cell-Free System ; Glutamic Acid* ; Heme Oxygenase-1 ; Membrane Potential, Mitochondrial ; Mitochondria ; Neurodegenerative Diseases ; Neurons ; Reactive Oxygen Species*

BACKGROUND: A primary central nervous system lymphoma (PCNSL) is a rare neoplasm with a poor prognosis. The treatment of PCNSL involves a combination of chemotherapy, intrathecal chemotherapy and radiotherapy. This study retrospectively evaluated the treatment outcomes and prognostic factors of Korean patients with PCNSL. METHODS: Between 1995 and 2003, 58 patients diagnosed with PCNSL from the multi-center hospitals were enrolled in this study. Among 56 patients who had received treatment, 16 patients were treated with radiotherapy alone, while 40 patients were treated with combined chemotherapy (CHOP; 9 cases, high-dose methotrexate; 31 cases) and radiotherapy. RESULTS: The median age of the patients was 58 years (range, 19-76). A diffuse large B-cell lymphoma was diagnosed in 56 cases (96.6%), while a peripheral T-cell lymphoma was diagnosed in 2 cases. Of the 47 patients who could be assessed for their response after treatment, a CR and PR was observed in 32 (68%) and 11 patients (23%), respectively, giving an overall response rate of 91% (95% CI, 82~100%). The estimated 3-year overall survival rate for all the patients was 67+/-7.9% and the 3-year disease free survival rate was 53+/-8.3%. The overall survival of the high-dose methotrexate group was superior to that of the CHOP group (77+/-10% versus 47+/-19%, p=0.05). Leukoencephalopathy was observed as a late complication in 9 patients (21%). No significant prognostic factors affecting survival were found by univariate analysis. CONCLUSIONS: Approximately half of the patients could have long-term survival after treatment in this study. High-dose methotrexate containing chemotherapy followed by radiotherapy was found to be an effective treatment.
Central Nervous System* ; Disease-Free Survival ; Drug Therapy ; Humans ; Leukoencephalopathies ; Lymphoma* ; Lymphoma, B-Cell ; Lymphoma, T-Cell, Peripheral ; Methotrexate ; Prognosis ; Radiotherapy ; Retrospective Studies* ; Survival Rate

Cell-free protein expression system is a new method to express target protein in vitro and has been widely applied to the study of protein structure, protein function and other related fields. Preparation of cell extract is one of the key factors that affect the efficiency of the cell-free system. To improve the efficiency and economical feasibility of cell-free protein synthesis, we discussed the parameters during the preparation of the cell extract. These parameters include centrifugation speed, pre-incubation, and dialysis. We used the green fluorescent protein as the reporter protein, and obtained a simple procedure for the preparation of Escherichia coli cell extract. A simple centrifugation step (12 000 x g, 10 min) followed by a brief incubation was sufficient for the preparation of an active cell extract to support protein expression with higher productivity (209 microg/mL). Compared to the traditional E. coli S30 procedure, the processing time was reduced by 62%, and the productivity was increased by 2.6 times. The new procedure will make the advantage of cell-free technology more obvious, and promote its wider application.
Cell Fractionation ; methods ; Cell-Free System ; Escherichia coli ; cytology ; genetics ; metabolism ; Escherichia coli Proteins ; biosynthesis ; chemistry ; isolation & purification ; Green Fluorescent Proteins ; metabolism