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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.


Related in: MedlinePlus

SDS-PAGE of steps in the purification of RFAP synthase from A. fulgidus. E. coli BL21(DE3) with pJWS1 was induced for the production of RFAP synthase encoded by the AF2089 gene. Samples were boiled for 10 min with reducing SDS-PAGE sample buffer prior to loading on a 12% polyacrylamide gel, and the gel was stained with Coomassie blue. Lane 1: uninduced cells (20 µg protein); lane 2: cells induced at 30˚C with 1 mM IPTG (20 µg protein); lane 3: cell-free extract (26 µg); lane 4: heated cell-free extract (27 µg); lane 5: hydroxyapatite fraction (17 µg); lane 6: MonoQ fraction (2 µg). The molecular mass markers (97.4, 66.2, 45, 31, 21.5, and 14 kilodaltons) are shown in lane 7. The protein encoded by AF2089 has a predicted molecular mass of 34 kilodaltons and is indicated by the arrow.
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Figure 3: SDS-PAGE of steps in the purification of RFAP synthase from A. fulgidus. E. coli BL21(DE3) with pJWS1 was induced for the production of RFAP synthase encoded by the AF2089 gene. Samples were boiled for 10 min with reducing SDS-PAGE sample buffer prior to loading on a 12% polyacrylamide gel, and the gel was stained with Coomassie blue. Lane 1: uninduced cells (20 µg protein); lane 2: cells induced at 30˚C with 1 mM IPTG (20 µg protein); lane 3: cell-free extract (26 µg); lane 4: heated cell-free extract (27 µg); lane 5: hydroxyapatite fraction (17 µg); lane 6: MonoQ fraction (2 µg). The molecular mass markers (97.4, 66.2, 45, 31, 21.5, and 14 kilodaltons) are shown in lane 7. The protein encoded by AF2089 has a predicted molecular mass of 34 kilodaltons and is indicated by the arrow.

Mentions: The RFAP synthase gene homolog from the hyperthermophilic archaeon A. fulgidus was previously designated as AF2089 (12). This gene was previously expressed in E. coli BL21(DE3) when the temperature for growth and induction was lowered from 37˚C to 30˚C (12). The lower temperature presumably slowed the rate of protein synthesis, enabling proteins to fold into an active conformation. For RFAP synthase from A. fulgidus, the use of an expression cell line containing rare codon tRNA synthetases did not facilitate the synthesis of active enzyme, but rather exacerbated the production of inclusion bodies (data not shown). Previous attempts to purify the enzyme using an 80˚C heat step followed by hydroxyapatite and Phenyl Sepharose column chromatography resulted in 95% pure protein with a specific activity of 290 nmol per min per mg protein at the temperature optimum of 70˚C. However, attempts to further purify the enzyme resulted in complete loss of activity (12). In the current work, we have purified AF2089 to homogeneity using a gentler 65˚C heat step followed by hydroxyapatite and anion exchange chromatography with linear gradients rather than step gradients (Fig. 3). The highest specific activity obtained for the purified fraction (240 nmol per min per mg protein) was comparable to the specific activity reported previously (12).


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 steps in the purification of RFAP synthase from A. fulgidus. E. coli BL21(DE3) with pJWS1 was induced for the production of RFAP synthase encoded by the AF2089 gene. Samples were boiled for 10 min with reducing SDS-PAGE sample buffer prior to loading on a 12% polyacrylamide gel, and the gel was stained with Coomassie blue. Lane 1: uninduced cells (20 µg protein); lane 2: cells induced at 30˚C with 1 mM IPTG (20 µg protein); lane 3: cell-free extract (26 µg); lane 4: heated cell-free extract (27 µg); lane 5: hydroxyapatite fraction (17 µg); lane 6: MonoQ fraction (2 µg). The molecular mass markers (97.4, 66.2, 45, 31, 21.5, and 14 kilodaltons) are shown in lane 7. The protein encoded by AF2089 has a predicted molecular mass of 34 kilodaltons and is indicated by the arrow.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC152576&req=5

Figure 3: SDS-PAGE of steps in the purification of RFAP synthase from A. fulgidus. E. coli BL21(DE3) with pJWS1 was induced for the production of RFAP synthase encoded by the AF2089 gene. Samples were boiled for 10 min with reducing SDS-PAGE sample buffer prior to loading on a 12% polyacrylamide gel, and the gel was stained with Coomassie blue. Lane 1: uninduced cells (20 µg protein); lane 2: cells induced at 30˚C with 1 mM IPTG (20 µg protein); lane 3: cell-free extract (26 µg); lane 4: heated cell-free extract (27 µg); lane 5: hydroxyapatite fraction (17 µg); lane 6: MonoQ fraction (2 µg). The molecular mass markers (97.4, 66.2, 45, 31, 21.5, and 14 kilodaltons) are shown in lane 7. The protein encoded by AF2089 has a predicted molecular mass of 34 kilodaltons and is indicated by the arrow.
Mentions: The RFAP synthase gene homolog from the hyperthermophilic archaeon A. fulgidus was previously designated as AF2089 (12). This gene was previously expressed in E. coli BL21(DE3) when the temperature for growth and induction was lowered from 37˚C to 30˚C (12). The lower temperature presumably slowed the rate of protein synthesis, enabling proteins to fold into an active conformation. For RFAP synthase from A. fulgidus, the use of an expression cell line containing rare codon tRNA synthetases did not facilitate the synthesis of active enzyme, but rather exacerbated the production of inclusion bodies (data not shown). Previous attempts to purify the enzyme using an 80˚C heat step followed by hydroxyapatite and Phenyl Sepharose column chromatography resulted in 95% pure protein with a specific activity of 290 nmol per min per mg protein at the temperature optimum of 70˚C. However, attempts to further purify the enzyme resulted in complete loss of activity (12). In the current work, we have purified AF2089 to homogeneity using a gentler 65˚C heat step followed by hydroxyapatite and anion exchange chromatography with linear gradients rather than step gradients (Fig. 3). The highest specific activity obtained for the purified fraction (240 nmol per min per mg protein) was comparable to the specific activity reported previously (12).

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