The Experts below are selected from a list of 171 Experts worldwide ranked by ideXlab platform

Toshiyuki Kohno - One of the best experts on this subject based on the ideXlab platform.

  • Production of proteins for NMR studies using the wheat germ Cell-Free System.
    Methods in molecular biology (Clifton N.J.), 2005
    Co-Authors: Toshiyuki Kohno
    Abstract:

    This chapter describes protocols for preparing 15N-labeled proteins (ubiquitin is used as an example) using Escherichia coli cells (with purification) and the wheat germ Cell-Free System (without purification). A comparison of 1H-15N heteronuclear single-quantum coherence (HSQC) spectra of yeast ubiquitin prepared using each method indicates that this wheat germ Cell-Free System may be used for rapid nuclear magnetic resonance analyses of proteins without purification.

  • A wheat germ Cell-Free System is a novel way to screen protein folding and function
    Protein Science, 2003
    Co-Authors: Eugene Hayato Morita, Tatsuya Sawasaki, Rikou Tanaka, Yaeta Endo, Toshiyuki Kohno
    Abstract:

    For high-throughput protein structural analysis, it is indispensable to develop a reliable protein overexpression System. Although many protein overexpression Systems, such as that involving Escherichia coli cells, have been developed, the number of overexpressed proteins showing the same biological activities as those of the native proteins is limited. A novel wheat germ Cell-Free protein synthesis System was developed recently, and most of the proteins functioning in solution were synthesized as soluble forms. This suggests the applicability of this protein synthesis method to determination of the solution structures of functional proteins. To examine this possibility, we have synthesized two 15N-labeled proteins and obtained 1H-15N HSQC spectra for them. The structural analysis of these proteins has already progressed with an E. coli overexpression System, and 1H-15N HSQC spectra for biologically active proteins have already been obtained. Comparing the spectra, we have shown that proteins synthesized with a wheat germ Cell-Free System have the proper protein folding and enough biological activity. This is the first experimental evidence of the applicability of the wheat germ Cell-Free protein synthesis System to high-throughput protein structural analysis.

Guy Ourisson - One of the best experts on this subject based on the ideXlab platform.

  • non specific lanosterol and hopanoid biosynthesis by a cell free System from the bacterium methylococcus capsulatus
    FEBS Journal, 2005
    Co-Authors: Michel Rohmer, P Bouvier, Guy Ourisson
    Abstract:

    : 1. A Cell-Free System from the bacterium Methylococcus capsulatus was incubated with [12-3H]-squalene; diploptene and diplopterol, normally present in the bacterium, were labelled. 2 The same Cell-Free System was incubated with (RS)-2,3-epoxy-2,3-dihydro-[3-3H]squalene. Several radioactive 3-hydroxytriterpenes were purifed. Lanosterol, which is normally present in this bacterium, was found labelled as well as 3-epilanosterol. In addition, radioactive 3 alpha-hydroxy and 3 beta-hydroxydiploptene were formed. 3. These data may be explained by the coexistence of two cyclases in M. capsulatus: a squalene/hopane cyclase and a squalene epoxide/lanosterol cyclase. The squalene cyclase exhibits the same lack of substrate specificity as those of Acetobacter pasteurianum and Tetrahymena pyriformis, i.e. in addition to its normal substrate squalene, it can cyclize the two enantiomers of squalene epoxide into 3-hydroxyhopanoids. 4. The presence of a squalene epoxide/lanosterol cyclase activity, which was suspected in view of the unique 3 beta-hydroxy 4 alpha-methyl steroids of M. capsulatus, was demonstrated by the labelling of lanosterol. More surprisingly 3-epilanosterol was also present and labelled. We showed that this does not derive from lanosterol by isomerization via a 3-oxo compound. Therefore the squalene expoxide cyclase of M. capsulatus, like the one of eukaryotes cyclizes the (3S) enantiomer of squalene epoxide into lanosterol. But it is definitely less substrate-specific as it can also cyclize the (3R) enantiomer into 3-epilanosterol.

  • non specific biosynthesis of hopane triterpenes by a cell free System from acetobacter pasteurianum
    FEBS Journal, 2005
    Co-Authors: Michel Rohmer, Claude Anding, Guy Ourisson
    Abstract:

    1. A Cell-Free System from the bacterium Acetobacter pasteurianum was incubated with [12-3H]squalene; diploptene and diplopterol, hopanoids normally present in the bacterium, were labelled. Their radioactivity was confirmed by purification using thin-layer chromatography, synthesis of derivatives and recrystallization to constant specific activity. This demonstrates the direct cyclization of squalene into diploptene and diplopterol, catalysed by a squalene cyclase activity in A. pasteurianum. 2. The same Cell-Free System transformed (RS)-2,3-epoxy-2,3-dihydro-[12,13-3H]squalene into labelled 3 alpha-hydroxyhop-22(29)-ene, 3 beta-hydroxyhop-22(29)-ene, hopane-3 alpha,22-diol and hopane-3 beta,22-diol. Their radioactivity was similarly confirmed. This bacterial homogenate is thus capable of cyclizing an unnatural substrate, 2,3-epoxy-squalene, into 3-hydroxyhopanoids normally absent in the bacterium. 3. The 3 alpha-hydroxy and 3 beta-hydroxyhopanoids could have been enzymatically interconverted via the 3-oxo compound. Synthetic racemic (RS)-2,3-epoxy-2,3-dihydro-[3-3H]squalene was incubated and gave rise to 3-3H-labelled 3 alpha and 3 beta-hydroxyhopanoids. This excludes an isomerization via a 3-oxo compound which would give unlabelled 3-hydroxyhopanoids. 4. In conclusion, the cyclase of A. pasteurianum accepts the replacement of the normal substrate, squalene, by the corresponding epoxide. Furthermore it is not selective in the stereochemistry of the epoxide and cyclizes both enantiomers, contrary to the epoxysqualene cyclase of eukaryotes.

Robert F. Bulleit - One of the best experts on this subject based on the ideXlab platform.

  • Functional protein expression from a DNA based wheat germ Cell-Free System
    Journal of Structural and Functional Genomics, 2007
    Co-Authors: Kate Qin Zhao, Robin Hurst, Michael R. Slater, Robert F. Bulleit
    Abstract:

    Wheat germ based eukaryotic Cell-Free Systems have been shown to be applicable for both functional and structural analyses of proteins. However, the existing methods might require specialized instrumentation and/or a separate mRNA synthesis step. We have developed a DNA based, highly productive, coupled transcription/translation wheat germ Cell-Free System that incorporates the normally separate mRNA synthesis step and does not require specialized instrumentation. Using a small-volume batch reaction with fluorescence labeling, DNA templates predicted to encode proteins could be quickly screened for their ability to direct the expression of proteins of the appropriate size. Protein yield can be increased as much as 2 to 4-fold in this System using a dialysis reaction, reaching ∼200–440 μg/ml in 10–20 h. Furthermore, enzyme activities can be assayed directly in the extract without further purification. Simple purification with affinity tags can be achieved in one-step and with minor modifications, efficient SeMet and [ U -^15N] labeling of >95% can be accomplished in this System. Thus, this efficient Cell-Free expression System can facilitate both functional and structural proteomics.

Michel Rohmer - One of the best experts on this subject based on the ideXlab platform.

  • non specific lanosterol and hopanoid biosynthesis by a cell free System from the bacterium methylococcus capsulatus
    FEBS Journal, 2005
    Co-Authors: Michel Rohmer, P Bouvier, Guy Ourisson
    Abstract:

    : 1. A Cell-Free System from the bacterium Methylococcus capsulatus was incubated with [12-3H]-squalene; diploptene and diplopterol, normally present in the bacterium, were labelled. 2 The same Cell-Free System was incubated with (RS)-2,3-epoxy-2,3-dihydro-[3-3H]squalene. Several radioactive 3-hydroxytriterpenes were purifed. Lanosterol, which is normally present in this bacterium, was found labelled as well as 3-epilanosterol. In addition, radioactive 3 alpha-hydroxy and 3 beta-hydroxydiploptene were formed. 3. These data may be explained by the coexistence of two cyclases in M. capsulatus: a squalene/hopane cyclase and a squalene epoxide/lanosterol cyclase. The squalene cyclase exhibits the same lack of substrate specificity as those of Acetobacter pasteurianum and Tetrahymena pyriformis, i.e. in addition to its normal substrate squalene, it can cyclize the two enantiomers of squalene epoxide into 3-hydroxyhopanoids. 4. The presence of a squalene epoxide/lanosterol cyclase activity, which was suspected in view of the unique 3 beta-hydroxy 4 alpha-methyl steroids of M. capsulatus, was demonstrated by the labelling of lanosterol. More surprisingly 3-epilanosterol was also present and labelled. We showed that this does not derive from lanosterol by isomerization via a 3-oxo compound. Therefore the squalene expoxide cyclase of M. capsulatus, like the one of eukaryotes cyclizes the (3S) enantiomer of squalene epoxide into lanosterol. But it is definitely less substrate-specific as it can also cyclize the (3R) enantiomer into 3-epilanosterol.

  • non specific biosynthesis of hopane triterpenes by a cell free System from acetobacter pasteurianum
    FEBS Journal, 2005
    Co-Authors: Michel Rohmer, Claude Anding, Guy Ourisson
    Abstract:

    1. A Cell-Free System from the bacterium Acetobacter pasteurianum was incubated with [12-3H]squalene; diploptene and diplopterol, hopanoids normally present in the bacterium, were labelled. Their radioactivity was confirmed by purification using thin-layer chromatography, synthesis of derivatives and recrystallization to constant specific activity. This demonstrates the direct cyclization of squalene into diploptene and diplopterol, catalysed by a squalene cyclase activity in A. pasteurianum. 2. The same Cell-Free System transformed (RS)-2,3-epoxy-2,3-dihydro-[12,13-3H]squalene into labelled 3 alpha-hydroxyhop-22(29)-ene, 3 beta-hydroxyhop-22(29)-ene, hopane-3 alpha,22-diol and hopane-3 beta,22-diol. Their radioactivity was similarly confirmed. This bacterial homogenate is thus capable of cyclizing an unnatural substrate, 2,3-epoxy-squalene, into 3-hydroxyhopanoids normally absent in the bacterium. 3. The 3 alpha-hydroxy and 3 beta-hydroxyhopanoids could have been enzymatically interconverted via the 3-oxo compound. Synthetic racemic (RS)-2,3-epoxy-2,3-dihydro-[3-3H]squalene was incubated and gave rise to 3-3H-labelled 3 alpha and 3 beta-hydroxyhopanoids. This excludes an isomerization via a 3-oxo compound which would give unlabelled 3-hydroxyhopanoids. 4. In conclusion, the cyclase of A. pasteurianum accepts the replacement of the normal substrate, squalene, by the corresponding epoxide. Furthermore it is not selective in the stereochemistry of the epoxide and cyclizes both enantiomers, contrary to the epoxysqualene cyclase of eukaryotes.

Eugene Hayato Morita - One of the best experts on this subject based on the ideXlab platform.

  • A wheat germ Cell-Free System is a novel way to screen protein folding and function
    Protein Science, 2003
    Co-Authors: Eugene Hayato Morita, Tatsuya Sawasaki, Rikou Tanaka, Yaeta Endo, Toshiyuki Kohno
    Abstract:

    For high-throughput protein structural analysis, it is indispensable to develop a reliable protein overexpression System. Although many protein overexpression Systems, such as that involving Escherichia coli cells, have been developed, the number of overexpressed proteins showing the same biological activities as those of the native proteins is limited. A novel wheat germ Cell-Free protein synthesis System was developed recently, and most of the proteins functioning in solution were synthesized as soluble forms. This suggests the applicability of this protein synthesis method to determination of the solution structures of functional proteins. To examine this possibility, we have synthesized two 15N-labeled proteins and obtained 1H-15N HSQC spectra for them. The structural analysis of these proteins has already progressed with an E. coli overexpression System, and 1H-15N HSQC spectra for biologically active proteins have already been obtained. Comparing the spectra, we have shown that proteins synthesized with a wheat germ Cell-Free System have the proper protein folding and enough biological activity. This is the first experimental evidence of the applicability of the wheat germ Cell-Free protein synthesis System to high-throughput protein structural analysis.