caption a7 protein erg10 cys91ala mutant source organism s cerevisiae  (ATCC)

Journal: Acta Crystallographica. Section F, Structural Biology Communications

Article Title: Crystal structure of cytoplasmic acetoacetyl-CoA thiolase from Saccharomyces cerevisiae

doi: 10.1107/S2053230X17016971

Figure Lengend Snippet: Structure solution and refinement

Article Snippet: The mutant protein was incubated at room temperature with CoA at a molar ratio of 1:10 for 1 h. Macromolecule-production information is summarized in Table 1 . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Protein ERG10 Cys91Ala mutant Source organism S. cerevisiae (strain ATCC 204508/S288c) S. cerevisiae (strain ATCC 204508/S288c) DNA source Genomic DNA Plasmid ERG10 in pET-22bN Forward primer † 5′-TTC CATATG TCTCAGAACGTTTAC-3′ 5′-AAGGTCTCTGCATCCGCTATGAAGGCAATC-3′ Reverse primer ‡ 5′-CCG CTCGAG TCATATCTTTTCAATGACAATAG-3′ 5′-TAGCGGATGCAGAGACCTTGTTAACTGTGC-3′ Cloning vector pET-22bN pET-22bN Expression vector pET-22bN pET-22bN Expression host E. coli BL21 (DE3) E. coli BL21 (DE3) Complete amino-acid sequence of the construct produced § MG HHHHHH GSHMSQNVYIVSTARTPIGSFQGSLSSKTAVELGAVALKGALAKVPELDASKDFDEIIFGNVLSANLGQAPARQVALAAGLSNHIVASTVNKVCASAMKAIILGAQSIKCGNADVVVAGGCESMTNAPYYMPAARAGAKFGQTVLVDGVERDGLNDAYDGLAMGVHAEKCARDWDITREQQDNFAIESYQKSQKSQKEGKFDNEIVPVTIKGFRGKPDTQVTKDEEPARLHVEKLRSARTVFQKENGTVTAANASPINDGAAAVILVSEKVLKEKNLKPLAIIKGWGEAAHQPADFTWAPSLAVPKALKHAGIEDINSVDYFEFNEAFSVVGLVNTKILKLDPSKVNVYGGAVALGHPLGCSGARVVVTLLSILQQEGGKIGVAAICNGGGGASSIVIEKI MG HHHHHH GSHMSQNVYIVSTARTPIGSFQGSLSSKTAVELGAVALKGALAKVPELDASKDFDEIIFGNVLSANLGQAPARQVALAAGLSNHIVASTVNKV A ASAMKAIILGAQSIKCGNADVVVAGGCESMTNAPYYMPAARAGAKFGQTVLVDGVERDGLNDAYDGLAMGVHAEKCARDWDITREQQDNFAIESYQKSQKSQKEGKFDNEIVPVTIKGFRGKPDTQVTKDEEPARLHVEKLRSARTVFQKENGTVTAANASPINDGAAAVILVSEKVLKEKNLKPLAIIKGWGEAAHQPADFTWAPSLAVPKALKHAGIEDINSVDYFEFNEAFSVVGLVNTKILKLDPSKVNVYGGAVALGHPLGCSGARVVVTLLSILQQEGGKIGVAAICNGGGGASSIVIEKI Open in a separate window † The NdeI site is underlined.

Techniques: Mutagenesis

Journal: Acta Crystallographica. Section F, Structural Biology Communications

Article Title: Crystal structure of cytoplasmic acetoacetyl-CoA thiolase from Saccharomyces cerevisiae

doi: 10.1107/S2053230X17016971

Figure Lengend Snippet: Macromolecule-production information

Article Snippet: The mutant protein was incubated at room temperature with CoA at a molar ratio of 1:10 for 1 h. Macromolecule-production information is summarized in Table 1 . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Protein ERG10 Cys91Ala mutant Source organism S. cerevisiae (strain ATCC 204508/S288c) S. cerevisiae (strain ATCC 204508/S288c) DNA source Genomic DNA Plasmid ERG10 in pET-22bN Forward primer † 5′-TTC CATATG TCTCAGAACGTTTAC-3′ 5′-AAGGTCTCTGCATCCGCTATGAAGGCAATC-3′ Reverse primer ‡ 5′-CCG CTCGAG TCATATCTTTTCAATGACAATAG-3′ 5′-TAGCGGATGCAGAGACCTTGTTAACTGTGC-3′ Cloning vector pET-22bN pET-22bN Expression vector pET-22bN pET-22bN Expression host E. coli BL21 (DE3) E. coli BL21 (DE3) Complete amino-acid sequence of the construct produced § MG HHHHHH GSHMSQNVYIVSTARTPIGSFQGSLSSKTAVELGAVALKGALAKVPELDASKDFDEIIFGNVLSANLGQAPARQVALAAGLSNHIVASTVNKVCASAMKAIILGAQSIKCGNADVVVAGGCESMTNAPYYMPAARAGAKFGQTVLVDGVERDGLNDAYDGLAMGVHAEKCARDWDITREQQDNFAIESYQKSQKSQKEGKFDNEIVPVTIKGFRGKPDTQVTKDEEPARLHVEKLRSARTVFQKENGTVTAANASPINDGAAAVILVSEKVLKEKNLKPLAIIKGWGEAAHQPADFTWAPSLAVPKALKHAGIEDINSVDYFEFNEAFSVVGLVNTKILKLDPSKVNVYGGAVALGHPLGCSGARVVVTLLSILQQEGGKIGVAAICNGGGGASSIVIEKI MG HHHHHH GSHMSQNVYIVSTARTPIGSFQGSLSSKTAVELGAVALKGALAKVPELDASKDFDEIIFGNVLSANLGQAPARQVALAAGLSNHIVASTVNKV A ASAMKAIILGAQSIKCGNADVVVAGGCESMTNAPYYMPAARAGAKFGQTVLVDGVERDGLNDAYDGLAMGVHAEKCARDWDITREQQDNFAIESYQKSQKSQKEGKFDNEIVPVTIKGFRGKPDTQVTKDEEPARLHVEKLRSARTVFQKENGTVTAANASPINDGAAAVILVSEKVLKEKNLKPLAIIKGWGEAAHQPADFTWAPSLAVPKALKHAGIEDINSVDYFEFNEAFSVVGLVNTKILKLDPSKVNVYGGAVALGHPLGCSGARVVVTLLSILQQEGGKIGVAAICNGGGGASSIVIEKI Open in a separate window † The NdeI site is underlined.

Techniques: Mutagenesis, Plasmid Preparation, Cloning, Expressing, Sequencing, Construct, Produced

Journal: Acta Crystallographica. Section F, Structural Biology Communications

Article Title: Crystal structure of cytoplasmic acetoacetyl-CoA thiolase from Saccharomyces cerevisiae

doi: 10.1107/S2053230X17016971

Figure Lengend Snippet: Crystallization

Article Snippet: The mutant protein was incubated at room temperature with CoA at a molar ratio of 1:10 for 1 h. Macromolecule-production information is summarized in Table 1 . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Protein ERG10 Cys91Ala mutant Source organism S. cerevisiae (strain ATCC 204508/S288c) S. cerevisiae (strain ATCC 204508/S288c) DNA source Genomic DNA Plasmid ERG10 in pET-22bN Forward primer † 5′-TTC CATATG TCTCAGAACGTTTAC-3′ 5′-AAGGTCTCTGCATCCGCTATGAAGGCAATC-3′ Reverse primer ‡ 5′-CCG CTCGAG TCATATCTTTTCAATGACAATAG-3′ 5′-TAGCGGATGCAGAGACCTTGTTAACTGTGC-3′ Cloning vector pET-22bN pET-22bN Expression vector pET-22bN pET-22bN Expression host E. coli BL21 (DE3) E. coli BL21 (DE3) Complete amino-acid sequence of the construct produced § MG HHHHHH GSHMSQNVYIVSTARTPIGSFQGSLSSKTAVELGAVALKGALAKVPELDASKDFDEIIFGNVLSANLGQAPARQVALAAGLSNHIVASTVNKVCASAMKAIILGAQSIKCGNADVVVAGGCESMTNAPYYMPAARAGAKFGQTVLVDGVERDGLNDAYDGLAMGVHAEKCARDWDITREQQDNFAIESYQKSQKSQKEGKFDNEIVPVTIKGFRGKPDTQVTKDEEPARLHVEKLRSARTVFQKENGTVTAANASPINDGAAAVILVSEKVLKEKNLKPLAIIKGWGEAAHQPADFTWAPSLAVPKALKHAGIEDINSVDYFEFNEAFSVVGLVNTKILKLDPSKVNVYGGAVALGHPLGCSGARVVVTLLSILQQEGGKIGVAAICNGGGGASSIVIEKI MG HHHHHH GSHMSQNVYIVSTARTPIGSFQGSLSSKTAVELGAVALKGALAKVPELDASKDFDEIIFGNVLSANLGQAPARQVALAAGLSNHIVASTVNKV A ASAMKAIILGAQSIKCGNADVVVAGGCESMTNAPYYMPAARAGAKFGQTVLVDGVERDGLNDAYDGLAMGVHAEKCARDWDITREQQDNFAIESYQKSQKSQKEGKFDNEIVPVTIKGFRGKPDTQVTKDEEPARLHVEKLRSARTVFQKENGTVTAANASPINDGAAAVILVSEKVLKEKNLKPLAIIKGWGEAAHQPADFTWAPSLAVPKALKHAGIEDINSVDYFEFNEAFSVVGLVNTKILKLDPSKVNVYGGAVALGHPLGCSGARVVVTLLSILQQEGGKIGVAAICNGGGGASSIVIEKI Open in a separate window † The NdeI site is underlined.

Techniques: Mutagenesis, Diffusion-based Assay, Protein Concentration

Journal: Acta Crystallographica. Section F, Structural Biology Communications

Article Title: Crystal structure of cytoplasmic acetoacetyl-CoA thiolase from Saccharomyces cerevisiae

doi: 10.1107/S2053230X17016971

Figure Lengend Snippet: Data collection and processing Values in parentheses are for the highest resolution shell.

Article Snippet: The mutant protein was incubated at room temperature with CoA at a molar ratio of 1:10 for 1 h. Macromolecule-production information is summarized in Table 1 . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Protein ERG10 Cys91Ala mutant Source organism S. cerevisiae (strain ATCC 204508/S288c) S. cerevisiae (strain ATCC 204508/S288c) DNA source Genomic DNA Plasmid ERG10 in pET-22bN Forward primer † 5′-TTC CATATG TCTCAGAACGTTTAC-3′ 5′-AAGGTCTCTGCATCCGCTATGAAGGCAATC-3′ Reverse primer ‡ 5′-CCG CTCGAG TCATATCTTTTCAATGACAATAG-3′ 5′-TAGCGGATGCAGAGACCTTGTTAACTGTGC-3′ Cloning vector pET-22bN pET-22bN Expression vector pET-22bN pET-22bN Expression host E. coli BL21 (DE3) E. coli BL21 (DE3) Complete amino-acid sequence of the construct produced § MG HHHHHH GSHMSQNVYIVSTARTPIGSFQGSLSSKTAVELGAVALKGALAKVPELDASKDFDEIIFGNVLSANLGQAPARQVALAAGLSNHIVASTVNKVCASAMKAIILGAQSIKCGNADVVVAGGCESMTNAPYYMPAARAGAKFGQTVLVDGVERDGLNDAYDGLAMGVHAEKCARDWDITREQQDNFAIESYQKSQKSQKEGKFDNEIVPVTIKGFRGKPDTQVTKDEEPARLHVEKLRSARTVFQKENGTVTAANASPINDGAAAVILVSEKVLKEKNLKPLAIIKGWGEAAHQPADFTWAPSLAVPKALKHAGIEDINSVDYFEFNEAFSVVGLVNTKILKLDPSKVNVYGGAVALGHPLGCSGARVVVTLLSILQQEGGKIGVAAICNGGGGASSIVIEKI MG HHHHHH GSHMSQNVYIVSTARTPIGSFQGSLSSKTAVELGAVALKGALAKVPELDASKDFDEIIFGNVLSANLGQAPARQVALAAGLSNHIVASTVNKV A ASAMKAIILGAQSIKCGNADVVVAGGCESMTNAPYYMPAARAGAKFGQTVLVDGVERDGLNDAYDGLAMGVHAEKCARDWDITREQQDNFAIESYQKSQKSQKEGKFDNEIVPVTIKGFRGKPDTQVTKDEEPARLHVEKLRSARTVFQKENGTVTAANASPINDGAAAVILVSEKVLKEKNLKPLAIIKGWGEAAHQPADFTWAPSLAVPKALKHAGIEDINSVDYFEFNEAFSVVGLVNTKILKLDPSKVNVYGGAVALGHPLGCSGARVVVTLLSILQQEGGKIGVAAICNGGGGASSIVIEKI Open in a separate window † The NdeI site is underlined.

Techniques: Mutagenesis

Journal: Acta Crystallographica. Section F, Structural Biology Communications

Article Title: Crystal structure of cytoplasmic acetoacetyl-CoA thiolase from Saccharomyces cerevisiae

doi: 10.1107/S2053230X17016971

Figure Lengend Snippet: Comparison of structural superpositions of molecule A (red or blue cartoon model) onto molecule B (green or purple cartoon model) of (a) ERG10 and (c) the Cys91Ala mutant. Polypeptide-chain movements occur at the N-terminus and C-terminus of the disordered region only in molecule A of ERG10, as indicated by the black and blue ellipses, respectively. The two corresponding regions in the Cys91Ala mutant with identical conformation are also indicated by black and blue ellipses, respectively. (b) A CoA molecule was docked into molecule A of ERG10 (red cartoon model) by superposing it on molecule A of T2 (cyan cartoon model; PDB entry 2ibu) in complex with a CoA molecule. His228 and Ser252 of molecule A of ERG10, which clash significantly with the docked CoA molecule, are highlighted with black circles.

Article Snippet: The mutant protein was incubated at room temperature with CoA at a molar ratio of 1:10 for 1 h. Macromolecule-production information is summarized in Table 1 . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Protein ERG10 Cys91Ala mutant Source organism S. cerevisiae (strain ATCC 204508/S288c) S. cerevisiae (strain ATCC 204508/S288c) DNA source Genomic DNA Plasmid ERG10 in pET-22bN Forward primer † 5′-TTC CATATG TCTCAGAACGTTTAC-3′ 5′-AAGGTCTCTGCATCCGCTATGAAGGCAATC-3′ Reverse primer ‡ 5′-CCG CTCGAG TCATATCTTTTCAATGACAATAG-3′ 5′-TAGCGGATGCAGAGACCTTGTTAACTGTGC-3′ Cloning vector pET-22bN pET-22bN Expression vector pET-22bN pET-22bN Expression host E. coli BL21 (DE3) E. coli BL21 (DE3) Complete amino-acid sequence of the construct produced § MG HHHHHH GSHMSQNVYIVSTARTPIGSFQGSLSSKTAVELGAVALKGALAKVPELDASKDFDEIIFGNVLSANLGQAPARQVALAAGLSNHIVASTVNKVCASAMKAIILGAQSIKCGNADVVVAGGCESMTNAPYYMPAARAGAKFGQTVLVDGVERDGLNDAYDGLAMGVHAEKCARDWDITREQQDNFAIESYQKSQKSQKEGKFDNEIVPVTIKGFRGKPDTQVTKDEEPARLHVEKLRSARTVFQKENGTVTAANASPINDGAAAVILVSEKVLKEKNLKPLAIIKGWGEAAHQPADFTWAPSLAVPKALKHAGIEDINSVDYFEFNEAFSVVGLVNTKILKLDPSKVNVYGGAVALGHPLGCSGARVVVTLLSILQQEGGKIGVAAICNGGGGASSIVIEKI MG HHHHHH GSHMSQNVYIVSTARTPIGSFQGSLSSKTAVELGAVALKGALAKVPELDASKDFDEIIFGNVLSANLGQAPARQVALAAGLSNHIVASTVNKV A ASAMKAIILGAQSIKCGNADVVVAGGCESMTNAPYYMPAARAGAKFGQTVLVDGVERDGLNDAYDGLAMGVHAEKCARDWDITREQQDNFAIESYQKSQKSQKEGKFDNEIVPVTIKGFRGKPDTQVTKDEEPARLHVEKLRSARTVFQKENGTVTAANASPINDGAAAVILVSEKVLKEKNLKPLAIIKGWGEAAHQPADFTWAPSLAVPKALKHAGIEDINSVDYFEFNEAFSVVGLVNTKILKLDPSKVNVYGGAVALGHPLGCSGARVVVTLLSILQQEGGKIGVAAICNGGGGASSIVIEKI Open in a separate window † The NdeI site is underlined.

Techniques: Comparison, Mutagenesis

Journal: Acta Crystallographica. Section F, Structural Biology Communications

Article Title: Crystal structure of cytoplasmic acetoacetyl-CoA thiolase from Saccharomyces cerevisiae

doi: 10.1107/S2053230X17016971

Figure Lengend Snippet: Tetrameric form of ERG10 in solution as analysed by analytical size-exclusion chromatography. The blue dots fitted with a green line correspond to protein standards of known molecular weights. The red dot indicates the target protein subjected to analysis.

Article Snippet: The mutant protein was incubated at room temperature with CoA at a molar ratio of 1:10 for 1 h. Macromolecule-production information is summarized in Table 1 . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Protein ERG10 Cys91Ala mutant Source organism S. cerevisiae (strain ATCC 204508/S288c) S. cerevisiae (strain ATCC 204508/S288c) DNA source Genomic DNA Plasmid ERG10 in pET-22bN Forward primer † 5′-TTC CATATG TCTCAGAACGTTTAC-3′ 5′-AAGGTCTCTGCATCCGCTATGAAGGCAATC-3′ Reverse primer ‡ 5′-CCG CTCGAG TCATATCTTTTCAATGACAATAG-3′ 5′-TAGCGGATGCAGAGACCTTGTTAACTGTGC-3′ Cloning vector pET-22bN pET-22bN Expression vector pET-22bN pET-22bN Expression host E. coli BL21 (DE3) E. coli BL21 (DE3) Complete amino-acid sequence of the construct produced § MG HHHHHH GSHMSQNVYIVSTARTPIGSFQGSLSSKTAVELGAVALKGALAKVPELDASKDFDEIIFGNVLSANLGQAPARQVALAAGLSNHIVASTVNKVCASAMKAIILGAQSIKCGNADVVVAGGCESMTNAPYYMPAARAGAKFGQTVLVDGVERDGLNDAYDGLAMGVHAEKCARDWDITREQQDNFAIESYQKSQKSQKEGKFDNEIVPVTIKGFRGKPDTQVTKDEEPARLHVEKLRSARTVFQKENGTVTAANASPINDGAAAVILVSEKVLKEKNLKPLAIIKGWGEAAHQPADFTWAPSLAVPKALKHAGIEDINSVDYFEFNEAFSVVGLVNTKILKLDPSKVNVYGGAVALGHPLGCSGARVVVTLLSILQQEGGKIGVAAICNGGGGASSIVIEKI MG HHHHHH GSHMSQNVYIVSTARTPIGSFQGSLSSKTAVELGAVALKGALAKVPELDASKDFDEIIFGNVLSANLGQAPARQVALAAGLSNHIVASTVNKV A ASAMKAIILGAQSIKCGNADVVVAGGCESMTNAPYYMPAARAGAKFGQTVLVDGVERDGLNDAYDGLAMGVHAEKCARDWDITREQQDNFAIESYQKSQKSQKEGKFDNEIVPVTIKGFRGKPDTQVTKDEEPARLHVEKLRSARTVFQKENGTVTAANASPINDGAAAVILVSEKVLKEKNLKPLAIIKGWGEAAHQPADFTWAPSLAVPKALKHAGIEDINSVDYFEFNEAFSVVGLVNTKILKLDPSKVNVYGGAVALGHPLGCSGARVVVTLLSILQQEGGKIGVAAICNGGGGASSIVIEKI Open in a separate window † The NdeI site is underlined.

Techniques: Size-exclusion Chromatography

Journal: Acta Crystallographica. Section F, Structural Biology Communications

Article Title: Crystal structure of cytoplasmic acetoacetyl-CoA thiolase from Saccharomyces cerevisiae

doi: 10.1107/S2053230X17016971

Figure Lengend Snippet: (a) Quaternary structure of the ERG10 tetramer generated by the dimerization of dimers. The tertiary structures of the four ERG10 monomers are distinguished by blue, green, magenta and cyan colours. (b) Overall cartoon representation of the ERG10 monomer. The N-terminal, C-terminal and loop domains are distinguished by dark blue, red and green colours, respectively. The covering loop is shown in purple and the tetramerization loop is shown in orange.

Article Snippet: The mutant protein was incubated at room temperature with CoA at a molar ratio of 1:10 for 1 h. Macromolecule-production information is summarized in Table 1 . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Protein ERG10 Cys91Ala mutant Source organism S. cerevisiae (strain ATCC 204508/S288c) S. cerevisiae (strain ATCC 204508/S288c) DNA source Genomic DNA Plasmid ERG10 in pET-22bN Forward primer † 5′-TTC CATATG TCTCAGAACGTTTAC-3′ 5′-AAGGTCTCTGCATCCGCTATGAAGGCAATC-3′ Reverse primer ‡ 5′-CCG CTCGAG TCATATCTTTTCAATGACAATAG-3′ 5′-TAGCGGATGCAGAGACCTTGTTAACTGTGC-3′ Cloning vector pET-22bN pET-22bN Expression vector pET-22bN pET-22bN Expression host E. coli BL21 (DE3) E. coli BL21 (DE3) Complete amino-acid sequence of the construct produced § MG HHHHHH GSHMSQNVYIVSTARTPIGSFQGSLSSKTAVELGAVALKGALAKVPELDASKDFDEIIFGNVLSANLGQAPARQVALAAGLSNHIVASTVNKVCASAMKAIILGAQSIKCGNADVVVAGGCESMTNAPYYMPAARAGAKFGQTVLVDGVERDGLNDAYDGLAMGVHAEKCARDWDITREQQDNFAIESYQKSQKSQKEGKFDNEIVPVTIKGFRGKPDTQVTKDEEPARLHVEKLRSARTVFQKENGTVTAANASPINDGAAAVILVSEKVLKEKNLKPLAIIKGWGEAAHQPADFTWAPSLAVPKALKHAGIEDINSVDYFEFNEAFSVVGLVNTKILKLDPSKVNVYGGAVALGHPLGCSGARVVVTLLSILQQEGGKIGVAAICNGGGGASSIVIEKI MG HHHHHH GSHMSQNVYIVSTARTPIGSFQGSLSSKTAVELGAVALKGALAKVPELDASKDFDEIIFGNVLSANLGQAPARQVALAAGLSNHIVASTVNKV A ASAMKAIILGAQSIKCGNADVVVAGGCESMTNAPYYMPAARAGAKFGQTVLVDGVERDGLNDAYDGLAMGVHAEKCARDWDITREQQDNFAIESYQKSQKSQKEGKFDNEIVPVTIKGFRGKPDTQVTKDEEPARLHVEKLRSARTVFQKENGTVTAANASPINDGAAAVILVSEKVLKEKNLKPLAIIKGWGEAAHQPADFTWAPSLAVPKALKHAGIEDINSVDYFEFNEAFSVVGLVNTKILKLDPSKVNVYGGAVALGHPLGCSGARVVVTLLSILQQEGGKIGVAAICNGGGGASSIVIEKI Open in a separate window † The NdeI site is underlined.

Techniques: Generated

Journal: Acta Crystallographica. Section F, Structural Biology Communications

Article Title: Crystal structure of cytoplasmic acetoacetyl-CoA thiolase from Saccharomyces cerevisiae

doi: 10.1107/S2053230X17016971

Figure Lengend Snippet: Surface-charge diagrams of (a) ERG10, (b) T2 and (c) bCT. Electrostatic surface potential diagrams were drawn in an identical orientation after superposition. The covering loops are shown as green, cyan and red stick models, respectively. His192 of T2, His156 of bCT and the corresponding Ala159 of ERG10 at the entrance to the pantetheine-binding tunnel are labelled. The negative potential of the covering loop, which is highlighted with a dotted ellipse, in ERG10 differs from the surface potential in T2 and bCT. The corresponding residues Glu156 (ERG10), Asn189 (T2) and Tyr153 (bCT) are also labelled.

Article Snippet: The mutant protein was incubated at room temperature with CoA at a molar ratio of 1:10 for 1 h. Macromolecule-production information is summarized in Table 1 . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Protein ERG10 Cys91Ala mutant Source organism S. cerevisiae (strain ATCC 204508/S288c) S. cerevisiae (strain ATCC 204508/S288c) DNA source Genomic DNA Plasmid ERG10 in pET-22bN Forward primer † 5′-TTC CATATG TCTCAGAACGTTTAC-3′ 5′-AAGGTCTCTGCATCCGCTATGAAGGCAATC-3′ Reverse primer ‡ 5′-CCG CTCGAG TCATATCTTTTCAATGACAATAG-3′ 5′-TAGCGGATGCAGAGACCTTGTTAACTGTGC-3′ Cloning vector pET-22bN pET-22bN Expression vector pET-22bN pET-22bN Expression host E. coli BL21 (DE3) E. coli BL21 (DE3) Complete amino-acid sequence of the construct produced § MG HHHHHH GSHMSQNVYIVSTARTPIGSFQGSLSSKTAVELGAVALKGALAKVPELDASKDFDEIIFGNVLSANLGQAPARQVALAAGLSNHIVASTVNKVCASAMKAIILGAQSIKCGNADVVVAGGCESMTNAPYYMPAARAGAKFGQTVLVDGVERDGLNDAYDGLAMGVHAEKCARDWDITREQQDNFAIESYQKSQKSQKEGKFDNEIVPVTIKGFRGKPDTQVTKDEEPARLHVEKLRSARTVFQKENGTVTAANASPINDGAAAVILVSEKVLKEKNLKPLAIIKGWGEAAHQPADFTWAPSLAVPKALKHAGIEDINSVDYFEFNEAFSVVGLVNTKILKLDPSKVNVYGGAVALGHPLGCSGARVVVTLLSILQQEGGKIGVAAICNGGGGASSIVIEKI MG HHHHHH GSHMSQNVYIVSTARTPIGSFQGSLSSKTAVELGAVALKGALAKVPELDASKDFDEIIFGNVLSANLGQAPARQVALAAGLSNHIVASTVNKV A ASAMKAIILGAQSIKCGNADVVVAGGCESMTNAPYYMPAARAGAKFGQTVLVDGVERDGLNDAYDGLAMGVHAEKCARDWDITREQQDNFAIESYQKSQKSQKEGKFDNEIVPVTIKGFRGKPDTQVTKDEEPARLHVEKLRSARTVFQKENGTVTAANASPINDGAAAVILVSEKVLKEKNLKPLAIIKGWGEAAHQPADFTWAPSLAVPKALKHAGIEDINSVDYFEFNEAFSVVGLVNTKILKLDPSKVNVYGGAVALGHPLGCSGARVVVTLLSILQQEGGKIGVAAICNGGGGASSIVIEKI Open in a separate window † The NdeI site is underlined.

Techniques: Binding Assay