Matsunaga S.,Yokohama City University |
Masaoka T.,National Hospital Organization Nagoya Medical Center |
Sawasaki T.,Ehime University |
Morishita R.,Yokohama City University |
And 10 more authors.
Frontiers in Microbiology | Year: 2015
Due to their high frequency of genomic mutations, human retroviruses often develop resistance to antiretroviral drugs. The emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) is a significant obstacle to the effective long-term treatment of HIV infection. The development of a rapid and versatile drug-susceptibility assay would enable acquisition of phenotypic information and facilitate determination of the appropriate choice of antiretroviral agents. In this study, we developed a novel in vitro method, termed the Cell-free drug susceptibility assay (CFDSA), for monitoring phenotypic information regarding the drug resistance of HIV-1 protease (PR). The CFDSA utilizes a wheat germ cell-free protein production system to synthesize enzymatically active HIV-1 PRs directly from PCR products amplified from HIV-1 molecular clones or clinical isolates in a rapid one-step procedure. Enzymatic activity of PRs can be readily measured by AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) in the presence or absence of clinically used protease inhibitors (PIs). CFDSA measurement of drug resistance was based on the fold resistance to the half-maximal inhibitory concentration (IC50) of various PIs. The CFDSA could serve as a non-infectious, rapid, accessible, and reliable alternative to infectious cell-based phenotypic assays for evaluation of PI-resistant HIV-1. © 2015 Matsunaga, Masaoka, Sawasaki, Morishita, Iwatani, Tatsumi, Endo, Yamamoto, Sugiura and Ryo.
PubMed | Osaka University, University of Tokyo, CellFree science Co., Hokkaido University and 4 more.
Type: Journal Article | Journal: Biochimica et biophysica acta | Year: 2016
Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8) has been investigated in molecular and biochemical detail, the physiological role of CASP8 is not fully understood. Here, we identified a two-pore domain potassium channel, tandem-pore domain halothane-inhibited K
Harbers M.,RIKEN |
Harbers M.,CellFree science Co.
FEBS Letters | Year: 2014
Cell-free protein expression plays an important role in biochemical research. However, only recent developments led to new methods to rapidly synthesize preparative amounts of protein that make cell-free protein expression an attractive alternative to cell-based methods. In particular the wheat germ system provides the highest translation efficiency among eukaryotic cell-free protein expression approaches and has a very high success rate for the expression of soluble proteins of good quality. As an open in vitro method, the wheat germ system is a preferable choice for many applications in protein research including options for protein labeling and the expression of difficult-to-express proteins like membrane proteins and multiple protein complexes. Here I describe wheat germ cell-free protein expression systems and give examples how they have been used in genome-wide expression studies, preparation of labeled proteins for structural genomics and protein mass spectroscopy, automated protein synthesis, and screening of enzymatic activities. Future directions for the use of cell-free expression methods are discussed. © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
PubMed | Abnova Taiwan Corporation, University of Toyama, Nagoya University, Hokkaido University and 2 more.
Type: | Journal: Scientific reports | Year: 2015
G-protein-coupled receptors (GPCRs) are one of the most important drug targets, and anti-GPCR monoclonal antibody (mAb) is an essential tool for functional analysis of GPCRs. However, it is very difficult to develop GPCR-specific mAbs due to difficulties in production of recombinant GPCR antigens, and lack of efficient mAb screening method. Here we describe a novel approach for the production of mAbs against GPCR using two original methods, bilayer-dialysis method and biotinylated liposome-based interaction assay (BiLIA), both of which are developed using wheat cell-free protein synthesis system and liposome technology. Using bilayer-dialysis method, various GPCRs were successfully synthesized with quality and quantity sufficient for immunization. For selection of specific mAb, we designed BiLIA that detects interaction between antibody and membrane protein on liposome. BiLIA prevented denaturation of GPCR, and then preferably selected conformation-sensitive antibodies. Using this approach, we successfully obtained mAbs against DRD1, GHSR, PTGER1 and T1R1. With respect to DRD1 mAb, 36 mouse mAbs and 6 rabbit mAbs were obtained which specifically recognized native DRD1 with high affinity. Among them, half of the mAbs were conformation-sensitive mAb, and two mAbs recognized extracellular loop 2 of DRD1. These results indicated that this approach is useful for GPCR mAb production.
Takemori N.,Ehime University |
Takemori A.,Ehime University |
Matsuoka K.,Ehime University |
Morishita R.,CellFree science Co. |
And 6 more authors.
Molecular BioSystems | Year: 2015
Using a wheat germ cell-free protein synthesis system, we developed a high-throughput method for the synthesis of stable isotope-labeled full-length transmembrane proteins as proteoliposomes to mimic the in vivo environment, and we successfully constructed an internal standard library for targeted transmembrane proteomics by using mass spectrometry. © The Royal Society of Chemistry 2015.
Madono M.,CellFree science Co. |
Sawasaki T.,Ehime University |
Morishita R.,CellFree science Co. |
Endo Y.,Ehime University
New Biotechnology | Year: 2011
Genomic information becomes useful knowledge only when the structures and functions of gene products are understood. In spite of a vast array of analytical tools developed for biological studies in recent years, producing proteins at will is still a bottleneck in post-genomic studies. The cell-free protein production system we developed using wheat embryos has enabled us to produce high quality proteins for genome-wide functional and structural analyses and at the same time circumvent almost all the limitations, such as biohazards and costs, that have hampered conventional cell-free protein synthesis systems. In the present article, we introduce examples of our new wheat germ cell-free protein production system and its application to functional and structural analyses, with the focus on the former. © 2010 Elsevier B.V.
Furuya Y.,Okayama University |
Denda M.,CellFree science Co. |
Sakane K.,Okayama University |
Ogusu T.,Okayama University |
And 5 more authors.
Cell Calcium | Year: 2016
To search for novel target(s) of the Ca2+-signaling transducer, calmodulin (CaM), we performed a newly developed genome-wide CaM interaction screening of 19,676 GST-fused proteins expressed in human. We identified striated muscle activator of Rho signaling (STARS) as a novel CaM target and characterized its CaM binding ability and found that the Ca2+/CaM complex interacted stoichiometrically with the N-terminal region (Ala13-Gln35) of STARS in vitro as well as in living cells. Mutagenesis studies identified Ile20 and Trp33 as the essential hydrophobic residues in CaM anchoring. Furthermore, the CaM binding deficient mutant (Ile20Ala, Trp33Ala) of STARS further enhanced its stimulatory effect on SRF-dependent transcriptional activation. These results suggest a connection between Ca2+-signaling via excitation-contraction coupling and the regulation of STARS-mediated gene expression in muscles. © 2016 Elsevier Ltd.
CellFree science Co. | Date: 2012-08-21
CELLFREE science CO. | Date: 2011-07-04
The present invention relates to a malaria vaccine comprising: (a) a polypeptide consisting of an amino acid sequence of SEQ ID NO: 1, 2, or 3; (b) a polypeptide consisting of an amino acid sequence of SEQ ID NO: 1, 2, or 3, wherein one or more amino acids are deleted, substituted and/or added and having effect for preventing falciparum malaria; or (c) a polypeptide consisting of an amino acid sequence having 70% or more identity with an amino acid sequence of SEQ ID NO: 1, 2, or 3 and having effect for preventing falciparum malaria.
PubMed | CellFree science Co. and Okayama University
Type: Journal Article | Journal: Cell calcium | Year: 2016
To search for novel target(s) of the Ca(2+)-signaling transducer, calmodulin (CaM), we performed a newly developed genome-wide CaM interaction screening of 19,676 GST-fused proteins expressed in human. We identified striated muscle activator of Rho signaling (STARS) as a novel CaM target and characterized its CaM binding ability and found that the Ca(2+)/CaM complex interacted stoichiometrically with the N-terminal region (Ala13-Gln35) of STARS in vitro as well as in living cells. Mutagenesis studies identified Ile20 and Trp33 as the essential hydrophobic residues in CaM anchoring. Furthermore, the CaM binding deficient mutant (Ile20Ala, Trp33Ala) of STARS further enhanced its stimulatory effect on SRF-dependent transcriptional activation. These results suggest a connection between Ca(2+)-signaling via excitation-contraction coupling and the regulation of STARS-mediated gene expression in muscles.