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Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli.

Bechard ME, Chhatwal S, Garcia RE, Rasche ME - Biol Proced Online (2003)

Bottom Line: By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity.The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone.This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Microbiology and Cell Science Department, University of Florida. Gainesville, FL 32611-0700. USA.

ABSTRACT
Tetrahydromethanopterin (H(4)MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H(4)MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H(4)MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase). Given the importance of RFAP synthase in H(4)MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H(4)MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

No MeSH data available.


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SDS-PAGE of soluble versus insoluble fractions of KB1 cells induced for the production of RFAP synthase from M. thermautotrophicus. KB1 cells were grown at 37˚C and then induced with IPTG at the indicated temperatures. Cells were broken with a French press and centrifuged, producing a soluble fraction (supernatant) and insoluble fraction (pellet). Induction at 37˚C for 2 h, soluble (lane 1) and insoluble (lane 2); induction at 30˚C for 6 h, soluble (lane 3) and insoluble (lane 4); induction at 20˚C for 16 h, soluble (lane 5) and insoluble (lane 6). Induction of chaperone, followed by induction of MTH0830 and incubation at 20˚C for 16 h, soluble (lane 7) and insoluble (lane 8). Each lane contained between 15 and 30 µg of protein. The arrow indicates the position of RFAP synthase from M. thermautotrophicus.
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Figure 5: SDS-PAGE of soluble versus insoluble fractions of KB1 cells induced for the production of RFAP synthase from M. thermautotrophicus. KB1 cells were grown at 37˚C and then induced with IPTG at the indicated temperatures. Cells were broken with a French press and centrifuged, producing a soluble fraction (supernatant) and insoluble fraction (pellet). Induction at 37˚C for 2 h, soluble (lane 1) and insoluble (lane 2); induction at 30˚C for 6 h, soluble (lane 3) and insoluble (lane 4); induction at 20˚C for 16 h, soluble (lane 5) and insoluble (lane 6). Induction of chaperone, followed by induction of MTH0830 and incubation at 20˚C for 16 h, soluble (lane 7) and insoluble (lane 8). Each lane contained between 15 and 30 µg of protein. The arrow indicates the position of RFAP synthase from M. thermautotrophicus.

Mentions: BLAST searches indicate that the gene MTH0830 from the methanogen M. thermautotrophicus is an RFAP synthase homolog (12). Initially, we attempted to express the gene by transforming E. coli BL21(DE3) cells with a plasmid (pED2) containing MTH0830. However, this procedure resulted in a complete lack of RFAP synthase activity even when the induction temperature was lowered to 30ºC (data not shown). This led us to create an expression strain (KB1) containing both pED2 and the plasmid pG-Tf2, which carries the genes for the GroEL/ES chaperone under the control of a tetracycline promoter (17). Fig. 5 illustrates the effects of induction temperature and the presence of chaperone on the production of soluble versus insoluble M. thermautotrophicus RFAP synthase.


Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli.

Bechard ME, Chhatwal S, Garcia RE, Rasche ME - Biol Proced Online (2003)

SDS-PAGE of soluble versus insoluble fractions of KB1 cells induced for the production of RFAP synthase from M. thermautotrophicus. KB1 cells were grown at 37˚C and then induced with IPTG at the indicated temperatures. Cells were broken with a French press and centrifuged, producing a soluble fraction (supernatant) and insoluble fraction (pellet). Induction at 37˚C for 2 h, soluble (lane 1) and insoluble (lane 2); induction at 30˚C for 6 h, soluble (lane 3) and insoluble (lane 4); induction at 20˚C for 16 h, soluble (lane 5) and insoluble (lane 6). Induction of chaperone, followed by induction of MTH0830 and incubation at 20˚C for 16 h, soluble (lane 7) and insoluble (lane 8). Each lane contained between 15 and 30 µg of protein. The arrow indicates the position of RFAP synthase from M. thermautotrophicus.
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Related In: Results  -  Collection

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Figure 5: SDS-PAGE of soluble versus insoluble fractions of KB1 cells induced for the production of RFAP synthase from M. thermautotrophicus. KB1 cells were grown at 37˚C and then induced with IPTG at the indicated temperatures. Cells were broken with a French press and centrifuged, producing a soluble fraction (supernatant) and insoluble fraction (pellet). Induction at 37˚C for 2 h, soluble (lane 1) and insoluble (lane 2); induction at 30˚C for 6 h, soluble (lane 3) and insoluble (lane 4); induction at 20˚C for 16 h, soluble (lane 5) and insoluble (lane 6). Induction of chaperone, followed by induction of MTH0830 and incubation at 20˚C for 16 h, soluble (lane 7) and insoluble (lane 8). Each lane contained between 15 and 30 µg of protein. The arrow indicates the position of RFAP synthase from M. thermautotrophicus.
Mentions: BLAST searches indicate that the gene MTH0830 from the methanogen M. thermautotrophicus is an RFAP synthase homolog (12). Initially, we attempted to express the gene by transforming E. coli BL21(DE3) cells with a plasmid (pED2) containing MTH0830. However, this procedure resulted in a complete lack of RFAP synthase activity even when the induction temperature was lowered to 30ºC (data not shown). This led us to create an expression strain (KB1) containing both pED2 and the plasmid pG-Tf2, which carries the genes for the GroEL/ES chaperone under the control of a tetracycline promoter (17). Fig. 5 illustrates the effects of induction temperature and the presence of chaperone on the production of soluble versus insoluble M. thermautotrophicus RFAP synthase.

Bottom Line: By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity.The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone.This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Microbiology and Cell Science Department, University of Florida. Gainesville, FL 32611-0700. USA.

ABSTRACT
Tetrahydromethanopterin (H(4)MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H(4)MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H(4)MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase). Given the importance of RFAP synthase in H(4)MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H(4)MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

No MeSH data available.


Related in: MedlinePlus