ndei Millipore Search Results


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  • 99
    New England Biolabs ndei
    Ndei, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 5999 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore ndei
    Cloning, expression, and purification of rPCNA. (A) Specific PCR of <t>PCNA.</t> Lane 1, 1-kb ladder; lanes 2 and 3, amplified PCR product at 882 bp. (B) Clone confirmation in pTZ57R/T. Lane 1, 1-kb ladder; lane 2; <t>NdeI-</t> and BamHI-digested pTZ57R/T-PCNA. (C)
    Ndei, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 14613 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore xhoi
    Cloning, expression, and purification of rPCNA. (A) Specific PCR of <t>PCNA.</t> Lane 1, 1-kb ladder; lanes 2 and 3, amplified PCR product at 882 bp. (B) Clone confirmation in pTZ57R/T. Lane 1, 1-kb ladder; lane 2; <t>NdeI-</t> and BamHI-digested pTZ57R/T-PCNA. (C)
    Xhoi, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5009 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bamhi
    Cloning, expression, and purification of rPCNA. (A) Specific PCR of <t>PCNA.</t> Lane 1, 1-kb ladder; lanes 2 and 3, amplified PCR product at 882 bp. (B) Clone confirmation in pTZ57R/T. Lane 1, 1-kb ladder; lane 2; <t>NdeI-</t> and BamHI-digested pTZ57R/T-PCNA. (C)
    Bamhi, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6614 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dna fragment
    DNase I footprinting analysis of YkrK binding to the <t>htpX</t> promoter region. (A) A BamHI-HindIII <t>DNA</t> fragment containing the htpX promoter region and spanning from positions −77 to +97 was labeled with 32 P at the HindIII site of the upper strand
    Dna Fragment, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4681 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore hindiii
    Combination of the origin components of plasmids SLP2, pSLA2, and SCP1 for replication. The PCR-amplified individual fragments (iterons or rep s of SLP2, pSLA2, and SCP1) (see text) were digested with restriction enzymes <t>HindIII</t> (abbreviated as Hi ) and
    Hindiii, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2925 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore e coli bl21
    Expression of P. gingivalis thiol peroxidase gene as purified recombinant protein. E. coli <t>BL21/pLysS</t> transformed with the expression vector pET17b containing the thiol peroxidase gene was grown and induced with IPTG. The cells were lysed, and the thiol
    E Coli Bl21, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10676 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore ecori
    Expression of P. gingivalis thiol peroxidase gene as purified recombinant protein. E. coli <t>BL21/pLysS</t> transformed with the expression vector pET17b containing the thiol peroxidase gene was grown and induced with IPTG. The cells were lysed, and the thiol
    Ecori, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3837 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore ncoi
    Domain architecture of <t>Rdh54</t> and rdh54 truncation mutants. A , schematic overview depicting the conserved regions of Rdh54, a 924-amino acid DNA translocase with a Rad51-binding domain of ∼100 amino acid ( aa ). The boxed regions represent the seven helicase-like Swi2/Snf2 motifs in Rdh54. The N-terminal truncations of Rdh54 used in this study are represented below. B , domain architecture of the Rad54-Rdh54 hybrid construct. The arrow represents an <t>NcoI</t> site used to fuse the Rad54 N-terminal domain with the C terminus of Rdh54.
    Ncoi, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3275 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bl21
    Domain architecture of <t>Rdh54</t> and rdh54 truncation mutants. A , schematic overview depicting the conserved regions of Rdh54, a 924-amino acid DNA translocase with a Rad51-binding domain of ∼100 amino acid ( aa ). The boxed regions represent the seven helicase-like Swi2/Snf2 motifs in Rdh54. The N-terminal truncations of Rdh54 used in this study are represented below. B , domain architecture of the Rad54-Rdh54 hybrid construct. The arrow represents an <t>NcoI</t> site used to fuse the Rad54 N-terminal domain with the C terminus of Rdh54.
    Bl21, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7940 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore pcr amplification
    Cell cycle progression and complementation of <t>cdkg1-2</t> . ( A ) Schematic representation of the CDKG1 locus and cdkg1-2 allele caused by insertion of a NIT1 plasmid and accompanying deletion (upper bracketed region). Coordinates are from genome assembly V5.5 taken from Phytozome ( http://phytozome.jgi.doe.gov ). Tall black rectangles represent exons and medium rectangles represent untranslated regions (UTR). Thin lines represent introns and intergenic regions. The lower bracketed region marks the 1.7kb 3’ UTR of CDKG1. Arrows represent the transcriptional starts and direction of transcription for URH1 and CDKG1 . Arrowheads mark binding sites for <t>PCR</t> primers used for genotyping: a1/b1 for CDKG1 last exon and adjacent 3’ UTR region, a4/b1 for NIT1 and CDKG1 3’ UTR junction (see Supplementary file 2 for detailed information). The 7 kb genomic region of the CDKG1 locus that was used to generate the HA-gCDKG1 complementation construct is shown below. The orange rectangle indicates the 3xHA tag located just downstream of the start codon. Primer sets a2/b2 and a3/b2 were used in RT-PCR experiments to amplify only HA-CDKG1 (a2/b2) or both endogenous and HA-CDKG1 cDNAs (a3/b2). ( B ) Ethidium bromide stained agarose gel showing genotyping PCR results for indicated strains. wt , wild-type; cdkg1-2, CDKG1 mutant strain; wt:HA-gCDKG1, transgenic line expressing HA-gCDKG1 ;. Results from representative non-complemented (1) and complemented (2, 3) progeny from a cross between cdkg1-2 and wt::HA-gCDKG1 are shown. Gene specific primer sets are shown on the right. Size phenotypes (large or wild type (WT)) are shown below. * Primer dimer. ( C ) Ethidium bromide stained agarose gel of RT-PCR products showing expression of HA-CDKG1 in a complemented cdkg1-2::HA-gCDKG1 strain. Strain genotypes are as described in panel ( B ). Gene specific amplicons and primer sets described in panel ( A ) are indicated to the right of each gel image. GBLP is an internal control. ( D ) Quantitative RT-PCR showing CDKG1 mRNA levels in synchronized wild-type ( WT ) or cdkg1-2::HA-gCDKG1 strains. All data were normalized to expression of internal control gene GBLP . Relative expression levels are shown based on values at time 0 hr that were set to 1. Error bars: S.D. of three replicates. ( E ) 12 hr light/12 hr dark synchronized wild type (black lines) and cdkg1-2 (orange lines) strains were monitored for passage through commitment (dashed lines), and for mitotic index (percentage in S/M phases) by light microscopy with fixed samples. The median cell size and time when the cultures had ~60% Committed cells is indicated by the dashed lines. DOI: http://dx.doi.org/10.7554/eLife.10767.004
    Pcr Amplification, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6447 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dna sequence
    The C-terminal tryptophan residue (W180) of <t>FAAP20</t> outside the compact UBZ module plays an indispensable role in ubiquitin recognition by FAAP20 in vitro and in efficient ICL <t>DNA</t> repair in vivo . ( A ) Measurement of binding affinity between WT FAAP20 and WT ubiquitin by ITC. ( B ) Mutation of W180A in FAAP20 abolished ubiquitin binding in ITC measurements. ( C ) U2OS cells stably expressing siRNA-resistant FAAP20 wild-type, C147A and C150A (CA), or W180A (WA) were transfected with siRNA against FAAP20 (siF20) for 48 h, and cell viability was determined 6 days after the treatment of indicated doses of mitomycin C. ( D ) Immunoblot analysis of U2OS cells in (C) harvested 72 h after siRNA treatment.
    Dna Sequence, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1775 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore e coli strain bl21
    The C-terminal tryptophan residue (W180) of <t>FAAP20</t> outside the compact UBZ module plays an indispensable role in ubiquitin recognition by FAAP20 in vitro and in efficient ICL <t>DNA</t> repair in vivo . ( A ) Measurement of binding affinity between WT FAAP20 and WT ubiquitin by ITC. ( B ) Mutation of W180A in FAAP20 abolished ubiquitin binding in ITC measurements. ( C ) U2OS cells stably expressing siRNA-resistant FAAP20 wild-type, C147A and C150A (CA), or W180A (WA) were transfected with siRNA against FAAP20 (siF20) for 48 h, and cell viability was determined 6 days after the treatment of indicated doses of mitomycin C. ( D ) Immunoblot analysis of U2OS cells in (C) harvested 72 h after siRNA treatment.
    E Coli Strain Bl21, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3521 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore expression vector pet 28a
    The C-terminal tryptophan residue (W180) of <t>FAAP20</t> outside the compact UBZ module plays an indispensable role in ubiquitin recognition by FAAP20 in vitro and in efficient ICL <t>DNA</t> repair in vivo . ( A ) Measurement of binding affinity between WT FAAP20 and WT ubiquitin by ITC. ( B ) Mutation of W180A in FAAP20 abolished ubiquitin binding in ITC measurements. ( C ) U2OS cells stably expressing siRNA-resistant FAAP20 wild-type, C147A and C150A (CA), or W180A (WA) were transfected with siRNA against FAAP20 (siF20) for 48 h, and cell viability was determined 6 days after the treatment of indicated doses of mitomycin C. ( D ) Immunoblot analysis of U2OS cells in (C) harvested 72 h after siRNA treatment.
    Expression Vector Pet 28a, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2077 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore iptg
    In vitro expression and enzymatic analysis of WspA protein. (a) Truncation of WspA1 for in vitro expression. (b) Induction of Wsp A and Wsp B in the E. coli BL21/pET28 expression system and their purification. P, total proteins in the absence of <t>IPTG</t> induction; IP, total proteins in the presence of IPTG induction; W1 and W2, washing fractions; E, elution fractions; D, dialyzed proteins; M, marker protein. (c) Blue-white test of transgenic E. coli cells on separate LB plates supplemented with X-Gal as the substrate. (d) In vitro activity analysis of purified Wsp A in X-Gal buffer (pH 7.5). (e) Comparative activity analysis in 0.1 M PBS buffer (pH 7.5) using ONPG as the substrate. Reactions were performed at <t>37°C.</t> Data shown are means ± SD ( n = 4). BSA, 50 μg/ml; Wsp A , 10 μg/ml; Wsp B , 27 μg/ml. Boiling conditions, 100°C for 15 min.
    Iptg, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12937 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore bglii
    In vitro expression and enzymatic analysis of WspA protein. (a) Truncation of WspA1 for in vitro expression. (b) Induction of Wsp A and Wsp B in the E. coli BL21/pET28 expression system and their purification. P, total proteins in the absence of <t>IPTG</t> induction; IP, total proteins in the presence of IPTG induction; W1 and W2, washing fractions; E, elution fractions; D, dialyzed proteins; M, marker protein. (c) Blue-white test of transgenic E. coli cells on separate LB plates supplemented with X-Gal as the substrate. (d) In vitro activity analysis of purified Wsp A in X-Gal buffer (pH 7.5). (e) Comparative activity analysis in 0.1 M PBS buffer (pH 7.5) using ONPG as the substrate. Reactions were performed at <t>37°C.</t> Data shown are means ± SD ( n = 4). BSA, 50 μg/ml; Wsp A , 10 μg/ml; Wsp B , 27 μg/ml. Boiling conditions, 100°C for 15 min.
    Bglii, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 530 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Cloning, expression, and purification of rPCNA. (A) Specific PCR of PCNA. Lane 1, 1-kb ladder; lanes 2 and 3, amplified PCR product at 882 bp. (B) Clone confirmation in pTZ57R/T. Lane 1, 1-kb ladder; lane 2; NdeI- and BamHI-digested pTZ57R/T-PCNA. (C)

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Characterization of the Proliferating Cell Nuclear Antigen of Leishmania donovani Clinical Isolates and Its Association with Antimony Resistance

    doi: 10.1128/AAC.01847-13

    Figure Lengend Snippet: Cloning, expression, and purification of rPCNA. (A) Specific PCR of PCNA. Lane 1, 1-kb ladder; lanes 2 and 3, amplified PCR product at 882 bp. (B) Clone confirmation in pTZ57R/T. Lane 1, 1-kb ladder; lane 2; NdeI- and BamHI-digested pTZ57R/T-PCNA. (C)

    Article Snippet: PCNA was further subcloned at the NdeI and BamHI sites in the bacterial expression vector pET28a (Novagen).

    Techniques: Clone Assay, Expressing, Purification, Polymerase Chain Reaction, Amplification

    Schematic representation of the pET-P T7 (RpoD), pET-P T7 (FliA), pACYC-P T7 (GFP), pACYC-P rrnB (GFP), and pACYC-P rrnB (Luc) plasmids. Ori, Escherichia coli replication origin; Kan r and Amp r , genes conferring resistance to kanamycin and ampicillin, respectively; RpoD, OY-M rpoD gene without its own promoter; FliA, OY-M fliA gene without its own promoter; Lac I, lactose repressor; P T7/lacI , T7 promoter regulated by Lac I; P rrnB , transcription promoter of the OY-M rrnB gene; GFP, gfp reporter gene; Luc, luciferase reporter gene; N, Nde I; S, Sal I; B, Bgl II; H, Hin dIII.

    Journal: MicrobiologyOpen

    Article Title: New ex vivo reporter assay system reveals that ? factors of an unculturable pathogen control gene regulation involved in the host switching between insects and plants

    doi: 10.1002/mbo3.93

    Figure Lengend Snippet: Schematic representation of the pET-P T7 (RpoD), pET-P T7 (FliA), pACYC-P T7 (GFP), pACYC-P rrnB (GFP), and pACYC-P rrnB (Luc) plasmids. Ori, Escherichia coli replication origin; Kan r and Amp r , genes conferring resistance to kanamycin and ampicillin, respectively; RpoD, OY-M rpoD gene without its own promoter; FliA, OY-M fliA gene without its own promoter; Lac I, lactose repressor; P T7/lacI , T7 promoter regulated by Lac I; P rrnB , transcription promoter of the OY-M rrnB gene; GFP, gfp reporter gene; Luc, luciferase reporter gene; N, Nde I; S, Sal I; B, Bgl II; H, Hin dIII.

    Article Snippet: The PCR product was digested with Nde I and Xho I restriction enzymes for rpoD , or Nde I and Hin dIII for fliA , and then cloned into the pET-30a vectors (Novagen, Madison, WI) through the same sites.

    Techniques: Positron Emission Tomography, Luciferase

    DNase I footprinting analysis of YkrK binding to the htpX promoter region. (A) A BamHI-HindIII DNA fragment containing the htpX promoter region and spanning from positions −77 to +97 was labeled with 32 P at the HindIII site of the upper strand

    Journal: Journal of Bacteriology

    Article Title: Reexamining Transcriptional Regulation of the Bacillus subtilis htpX Gene and the ykrK Gene, Encoding a Novel Type of Transcriptional Regulator, and Redefining the YkrK Operator

    doi: 10.1128/JB.01258-12

    Figure Lengend Snippet: DNase I footprinting analysis of YkrK binding to the htpX promoter region. (A) A BamHI-HindIII DNA fragment containing the htpX promoter region and spanning from positions −77 to +97 was labeled with 32 P at the HindIII site of the upper strand

    Article Snippet: To construct plasmid pGS2264 for overproduction of His-tagged HtpX in E. coli , a DNA fragment carrying the coding sequence of htpX was amplified by PCR using primer A569 plus A570 and was then ligated into NdeI- and XhoI-digested pET22b (Novagen).

    Techniques: Footprinting, Binding Assay, Labeling

    EMSAs of the interaction of YkrK with various DNA probes. A 32 P-labeled DNA fragment containing the wild-type inverted repeat in the htpX promoter region and spanning from positions −77 to +97 (relative to the transcriptional initiation site of

    Journal: Journal of Bacteriology

    Article Title: Reexamining Transcriptional Regulation of the Bacillus subtilis htpX Gene and the ykrK Gene, Encoding a Novel Type of Transcriptional Regulator, and Redefining the YkrK Operator

    doi: 10.1128/JB.01258-12

    Figure Lengend Snippet: EMSAs of the interaction of YkrK with various DNA probes. A 32 P-labeled DNA fragment containing the wild-type inverted repeat in the htpX promoter region and spanning from positions −77 to +97 (relative to the transcriptional initiation site of

    Article Snippet: To construct plasmid pGS2264 for overproduction of His-tagged HtpX in E. coli , a DNA fragment carrying the coding sequence of htpX was amplified by PCR using primer A569 plus A570 and was then ligated into NdeI- and XhoI-digested pET22b (Novagen).

    Techniques: Labeling

    Vector construction and protein overproduction. ( A ) Construction of the bicistronic and two-promoter vectors. The vectors used for producing the BCL-X L :BAD complex is presented as a representative. T7, T5, and TEV denote T7 promoter, T5 promoter, and TEV protease cleavage site, respectively. The lines with double-arrows indicate the amplified DNA fragments used to produce the bicistronic or the two-promoter expression vector. ( B ) SDS-PAGE analysis. The production levels of the BCL-X L :BAD, BCL-X L :BIM-S, and CED-9:EGL-1 complexes were analyzed. Cleared cell lysate (C) and supernatant of the cell lysate (S) were loaded on a denaturing gel. Protein samples partially purified using a Ni-NTA column are denoted by “N.” ( C ) Quantitative representation of protein production. “Total expressed protein” indicates the partially purified proteins from a Ni-NTA column as in b , which was the mixture of VHb-BCL-X L or VHb-CED-9 and the protein in complex with the binding partner. “Protein complex” indicates the purified complexes prepared by removing the excessive uncomplexed VHb-BCL-X L or VHb-CED-9 from the N fraction using an anion exchange column and, when needed, a Superdex G75 gel filtration column. “BC” and “TP” indicate the bicistronic and the two-promoter expression system, respectively. All data represent means ± standard deviation (error bars) of three separate experiments.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Two-promoter vector is highly efficient for overproduction of protein complexes

    doi: 10.1110/ps.04644504

    Figure Lengend Snippet: Vector construction and protein overproduction. ( A ) Construction of the bicistronic and two-promoter vectors. The vectors used for producing the BCL-X L :BAD complex is presented as a representative. T7, T5, and TEV denote T7 promoter, T5 promoter, and TEV protease cleavage site, respectively. The lines with double-arrows indicate the amplified DNA fragments used to produce the bicistronic or the two-promoter expression vector. ( B ) SDS-PAGE analysis. The production levels of the BCL-X L :BAD, BCL-X L :BIM-S, and CED-9:EGL-1 complexes were analyzed. Cleared cell lysate (C) and supernatant of the cell lysate (S) were loaded on a denaturing gel. Protein samples partially purified using a Ni-NTA column are denoted by “N.” ( C ) Quantitative representation of protein production. “Total expressed protein” indicates the partially purified proteins from a Ni-NTA column as in b , which was the mixture of VHb-BCL-X L or VHb-CED-9 and the protein in complex with the binding partner. “Protein complex” indicates the purified complexes prepared by removing the excessive uncomplexed VHb-BCL-X L or VHb-CED-9 from the N fraction using an anion exchange column and, when needed, a Superdex G75 gel filtration column. “BC” and “TP” indicate the bicistronic and the two-promoter expression system, respectively. All data represent means ± standard deviation (error bars) of three separate experiments.

    Article Snippet: DNA fragments coding for BIM-S and BAD were amplified from the same library and ligated into the NdeI and XhoI restriction sites of the pET30a plasmid (Novagen).

    Techniques: Plasmid Preparation, Amplification, Expressing, SDS Page, Purification, Binding Assay, Filtration, Standard Deviation

    Combination of the origin components of plasmids SLP2, pSLA2, and SCP1 for replication. The PCR-amplified individual fragments (iterons or rep s of SLP2, pSLA2, and SCP1) (see text) were digested with restriction enzymes HindIII (abbreviated as Hi ) and

    Journal:

    Article Title: Characterization of the Genetic Components of Streptomyces lividans Linear Plasmid SLP2 for Replication in Circular and Linear Modes

    doi: 10.1128/JB.00873-06

    Figure Lengend Snippet: Combination of the origin components of plasmids SLP2, pSLA2, and SCP1 for replication. The PCR-amplified individual fragments (iterons or rep s of SLP2, pSLA2, and SCP1) (see text) were digested with restriction enzymes HindIII (abbreviated as Hi ) and

    Article Snippet: The ilrA SLP2 gene was cloned into the NdeI and HindIII sites of E. coli plasmid pET28b (Novagen) to obtain pXQ165 and introduced into E. coli strain BL21(DE3) containing plasmid pQC630 (Z. Qin and S. N. Cohen, unpublished), which was constructed by ligating pACYC184 with the tRNA genes for the rarely used arginine and proline codons.

    Techniques: Polymerase Chain Reaction, Amplification

    Expression of P. gingivalis thiol peroxidase gene as purified recombinant protein. E. coli BL21/pLysS transformed with the expression vector pET17b containing the thiol peroxidase gene was grown and induced with IPTG. The cells were lysed, and the thiol

    Journal:

    Article Title: T-Cell Expression Cloning of Porphyromonas gingivalis Genes Coding for T Helper-Biased Immune Responses during Infection

    doi: 10.1128/IAI.02029-05

    Figure Lengend Snippet: Expression of P. gingivalis thiol peroxidase gene as purified recombinant protein. E. coli BL21/pLysS transformed with the expression vector pET17b containing the thiol peroxidase gene was grown and induced with IPTG. The cells were lysed, and the thiol

    Article Snippet: Ligation products were subsequently transformed into E. coli BL21(DE3)/pLysS host cells (Novagen) for expression.

    Techniques: Expressing, Purification, Recombinant, Transformation Assay, Plasmid Preparation

    Domain architecture of Rdh54 and rdh54 truncation mutants. A , schematic overview depicting the conserved regions of Rdh54, a 924-amino acid DNA translocase with a Rad51-binding domain of ∼100 amino acid ( aa ). The boxed regions represent the seven helicase-like Swi2/Snf2 motifs in Rdh54. The N-terminal truncations of Rdh54 used in this study are represented below. B , domain architecture of the Rad54-Rdh54 hybrid construct. The arrow represents an NcoI site used to fuse the Rad54 N-terminal domain with the C terminus of Rdh54.

    Journal: The Journal of Biological Chemistry

    Article Title: Characterization of the Interaction between the Saccharomyces cerevisiae Rad51 Recombinase and the DNA Translocase Rdh54

    doi: 10.1074/jbc.M113.480475

    Figure Lengend Snippet: Domain architecture of Rdh54 and rdh54 truncation mutants. A , schematic overview depicting the conserved regions of Rdh54, a 924-amino acid DNA translocase with a Rad51-binding domain of ∼100 amino acid ( aa ). The boxed regions represent the seven helicase-like Swi2/Snf2 motifs in Rdh54. The N-terminal truncations of Rdh54 used in this study are represented below. B , domain architecture of the Rad54-Rdh54 hybrid construct. The arrow represents an NcoI site used to fuse the Rad54 N-terminal domain with the C terminus of Rdh54.

    Article Snippet: DNA plasmids for bacterial expression and protein purification were generated by PCR amplification and cloning of wild-type RDH54 , rdh54 truncation alleles, and RAD54-RDH54 hybrid sequence into the NcoI and XhoI sites of the pET32a vector (Novagen) to add thioredoxin and His6 tags to the N terminus of each protein.

    Techniques: Binding Assay, Construct

    Cell cycle progression and complementation of cdkg1-2 . ( A ) Schematic representation of the CDKG1 locus and cdkg1-2 allele caused by insertion of a NIT1 plasmid and accompanying deletion (upper bracketed region). Coordinates are from genome assembly V5.5 taken from Phytozome ( http://phytozome.jgi.doe.gov ). Tall black rectangles represent exons and medium rectangles represent untranslated regions (UTR). Thin lines represent introns and intergenic regions. The lower bracketed region marks the 1.7kb 3’ UTR of CDKG1. Arrows represent the transcriptional starts and direction of transcription for URH1 and CDKG1 . Arrowheads mark binding sites for PCR primers used for genotyping: a1/b1 for CDKG1 last exon and adjacent 3’ UTR region, a4/b1 for NIT1 and CDKG1 3’ UTR junction (see Supplementary file 2 for detailed information). The 7 kb genomic region of the CDKG1 locus that was used to generate the HA-gCDKG1 complementation construct is shown below. The orange rectangle indicates the 3xHA tag located just downstream of the start codon. Primer sets a2/b2 and a3/b2 were used in RT-PCR experiments to amplify only HA-CDKG1 (a2/b2) or both endogenous and HA-CDKG1 cDNAs (a3/b2). ( B ) Ethidium bromide stained agarose gel showing genotyping PCR results for indicated strains. wt , wild-type; cdkg1-2, CDKG1 mutant strain; wt:HA-gCDKG1, transgenic line expressing HA-gCDKG1 ;. Results from representative non-complemented (1) and complemented (2, 3) progeny from a cross between cdkg1-2 and wt::HA-gCDKG1 are shown. Gene specific primer sets are shown on the right. Size phenotypes (large or wild type (WT)) are shown below. * Primer dimer. ( C ) Ethidium bromide stained agarose gel of RT-PCR products showing expression of HA-CDKG1 in a complemented cdkg1-2::HA-gCDKG1 strain. Strain genotypes are as described in panel ( B ). Gene specific amplicons and primer sets described in panel ( A ) are indicated to the right of each gel image. GBLP is an internal control. ( D ) Quantitative RT-PCR showing CDKG1 mRNA levels in synchronized wild-type ( WT ) or cdkg1-2::HA-gCDKG1 strains. All data were normalized to expression of internal control gene GBLP . Relative expression levels are shown based on values at time 0 hr that were set to 1. Error bars: S.D. of three replicates. ( E ) 12 hr light/12 hr dark synchronized wild type (black lines) and cdkg1-2 (orange lines) strains were monitored for passage through commitment (dashed lines), and for mitotic index (percentage in S/M phases) by light microscopy with fixed samples. The median cell size and time when the cultures had ~60% Committed cells is indicated by the dashed lines. DOI: http://dx.doi.org/10.7554/eLife.10767.004

    Journal: eLife

    Article Title: A new class of cyclin dependent kinase in Chlamydomonas is required for coupling cell size to cell division

    doi: 10.7554/eLife.10767

    Figure Lengend Snippet: Cell cycle progression and complementation of cdkg1-2 . ( A ) Schematic representation of the CDKG1 locus and cdkg1-2 allele caused by insertion of a NIT1 plasmid and accompanying deletion (upper bracketed region). Coordinates are from genome assembly V5.5 taken from Phytozome ( http://phytozome.jgi.doe.gov ). Tall black rectangles represent exons and medium rectangles represent untranslated regions (UTR). Thin lines represent introns and intergenic regions. The lower bracketed region marks the 1.7kb 3’ UTR of CDKG1. Arrows represent the transcriptional starts and direction of transcription for URH1 and CDKG1 . Arrowheads mark binding sites for PCR primers used for genotyping: a1/b1 for CDKG1 last exon and adjacent 3’ UTR region, a4/b1 for NIT1 and CDKG1 3’ UTR junction (see Supplementary file 2 for detailed information). The 7 kb genomic region of the CDKG1 locus that was used to generate the HA-gCDKG1 complementation construct is shown below. The orange rectangle indicates the 3xHA tag located just downstream of the start codon. Primer sets a2/b2 and a3/b2 were used in RT-PCR experiments to amplify only HA-CDKG1 (a2/b2) or both endogenous and HA-CDKG1 cDNAs (a3/b2). ( B ) Ethidium bromide stained agarose gel showing genotyping PCR results for indicated strains. wt , wild-type; cdkg1-2, CDKG1 mutant strain; wt:HA-gCDKG1, transgenic line expressing HA-gCDKG1 ;. Results from representative non-complemented (1) and complemented (2, 3) progeny from a cross between cdkg1-2 and wt::HA-gCDKG1 are shown. Gene specific primer sets are shown on the right. Size phenotypes (large or wild type (WT)) are shown below. * Primer dimer. ( C ) Ethidium bromide stained agarose gel of RT-PCR products showing expression of HA-CDKG1 in a complemented cdkg1-2::HA-gCDKG1 strain. Strain genotypes are as described in panel ( B ). Gene specific amplicons and primer sets described in panel ( A ) are indicated to the right of each gel image. GBLP is an internal control. ( D ) Quantitative RT-PCR showing CDKG1 mRNA levels in synchronized wild-type ( WT ) or cdkg1-2::HA-gCDKG1 strains. All data were normalized to expression of internal control gene GBLP . Relative expression levels are shown based on values at time 0 hr that were set to 1. Error bars: S.D. of three replicates. ( E ) 12 hr light/12 hr dark synchronized wild type (black lines) and cdkg1-2 (orange lines) strains were monitored for passage through commitment (dashed lines), and for mitotic index (percentage in S/M phases) by light microscopy with fixed samples. The median cell size and time when the cultures had ~60% Committed cells is indicated by the dashed lines. DOI: http://dx.doi.org/10.7554/eLife.10767.004

    Article Snippet: CDKG1 antibody generation pET28a-CDKG1 or pET28a-CDKG1∆N (missing residues 1–92) were made by PCR amplification from pGEM-T-CDKG1 using primer sets described in and ligation into pET28a (EMD Millipore, Billerica, MA) at NdeI/EcoRI sites. pGST-MAT3 was generated as described previously ( ).

    Techniques: Plasmid Preparation, Binding Assay, Polymerase Chain Reaction, Construct, Reverse Transcription Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Mutagenesis, Transgenic Assay, Expressing, Quantitative RT-PCR, Light Microscopy

    Expression profiles and interactions between D cyclins, CDKG1 and MAT3/RBR during the cell cycle. ( A ) Ethidium bromide stained agarose gels showing amplification of cDNAs made with RNA samples taken from synchronized cultures in a 14 hr light/10 hr dark cycle at four stages: daughter cells (1 hr light), post-commitment cells (10 hr light), S/M phase cells (1 hr dark) and post-mitotic cells (4 hr dark). Primers used are listed in Supplementary file 2 and the number of PCR amplification cycles used for each reaction is displayed on the right. GBLP is an internal control. ( B ) Profiles of CDKG1, CDKB1 and GBLP mRNAs as described in ( A ), except the pre-commitment sample was from 4 hr light. ( C ) Interaction of CYCD3 AxAxA mutant with CDKG1 and MAT3. Mutated CYCD3 (LxCxE→AxAxA) was fused to the Gal4 activation domain (AD) and tested in an Y2H assay with MAT3/RBR and CDKG1. Empty indicates vector-only with no fusion protein. Growth of two independent co-transformants is shown, and the relative strength of interaction is indicated by -, no interaction, +, weak interaction, and +++, very strong interaction. ( D ) Bar graph shows relative kinase activity of IVT CDKG1, CDKG1 kd and CDKG1+CYCD3 using GST-MAT3 as a substrate. The amount of 32 P-labeled GST-MAT3 from each reaction was normalized to the value from lane 2. Data are expressed as the mean of three independent experiments. Error bar: S.D. ( E ) Immunoprecipitation (IP) from whole cell extracts, fractionation by SDS-PAGE and detection of CDKG1 by Western blotting using polyclonal antisera raised against full length CDGK1. All strains used were synchronized in S/M phase. Lanes 1,2 are concentrated input fractions prior to IP from indicated strains. Anti-HA (lanes 3,4) or anti-CDKG1 (lanes 5–8) antibodies were used for IPs as indicated and the supernatant (lanes 3,5,7) or pellet (lanes 4,6,8) fractions probed using anti-CDKG1. The anti-CDKG1 antibody detects a single band near the predicted molecular weight of 44 kDa that is not present in cdkg1-2 mutants. ( F ) Schematic of the regulatory hierarchy for the Chlamydomonas mitotic sizer pathway (left side) and the metazoan G1 control pathway (right side). Both pathways integrate internal and/or external signals for cell cycle progression through D-cyclin dependent CDKs that phosphorylate RB-related proteins. DOI: http://dx.doi.org/10.7554/eLife.10767.009

    Journal: eLife

    Article Title: A new class of cyclin dependent kinase in Chlamydomonas is required for coupling cell size to cell division

    doi: 10.7554/eLife.10767

    Figure Lengend Snippet: Expression profiles and interactions between D cyclins, CDKG1 and MAT3/RBR during the cell cycle. ( A ) Ethidium bromide stained agarose gels showing amplification of cDNAs made with RNA samples taken from synchronized cultures in a 14 hr light/10 hr dark cycle at four stages: daughter cells (1 hr light), post-commitment cells (10 hr light), S/M phase cells (1 hr dark) and post-mitotic cells (4 hr dark). Primers used are listed in Supplementary file 2 and the number of PCR amplification cycles used for each reaction is displayed on the right. GBLP is an internal control. ( B ) Profiles of CDKG1, CDKB1 and GBLP mRNAs as described in ( A ), except the pre-commitment sample was from 4 hr light. ( C ) Interaction of CYCD3 AxAxA mutant with CDKG1 and MAT3. Mutated CYCD3 (LxCxE→AxAxA) was fused to the Gal4 activation domain (AD) and tested in an Y2H assay with MAT3/RBR and CDKG1. Empty indicates vector-only with no fusion protein. Growth of two independent co-transformants is shown, and the relative strength of interaction is indicated by -, no interaction, +, weak interaction, and +++, very strong interaction. ( D ) Bar graph shows relative kinase activity of IVT CDKG1, CDKG1 kd and CDKG1+CYCD3 using GST-MAT3 as a substrate. The amount of 32 P-labeled GST-MAT3 from each reaction was normalized to the value from lane 2. Data are expressed as the mean of three independent experiments. Error bar: S.D. ( E ) Immunoprecipitation (IP) from whole cell extracts, fractionation by SDS-PAGE and detection of CDKG1 by Western blotting using polyclonal antisera raised against full length CDGK1. All strains used were synchronized in S/M phase. Lanes 1,2 are concentrated input fractions prior to IP from indicated strains. Anti-HA (lanes 3,4) or anti-CDKG1 (lanes 5–8) antibodies were used for IPs as indicated and the supernatant (lanes 3,5,7) or pellet (lanes 4,6,8) fractions probed using anti-CDKG1. The anti-CDKG1 antibody detects a single band near the predicted molecular weight of 44 kDa that is not present in cdkg1-2 mutants. ( F ) Schematic of the regulatory hierarchy for the Chlamydomonas mitotic sizer pathway (left side) and the metazoan G1 control pathway (right side). Both pathways integrate internal and/or external signals for cell cycle progression through D-cyclin dependent CDKs that phosphorylate RB-related proteins. DOI: http://dx.doi.org/10.7554/eLife.10767.009

    Article Snippet: CDKG1 antibody generation pET28a-CDKG1 or pET28a-CDKG1∆N (missing residues 1–92) were made by PCR amplification from pGEM-T-CDKG1 using primer sets described in and ligation into pET28a (EMD Millipore, Billerica, MA) at NdeI/EcoRI sites. pGST-MAT3 was generated as described previously ( ).

    Techniques: Expressing, Staining, Amplification, Polymerase Chain Reaction, Mutagenesis, Activation Assay, Y2H Assay, Plasmid Preparation, Activity Assay, Labeling, Immunoprecipitation, Fractionation, SDS Page, Western Blot, Molecular Weight

    The C-terminal tryptophan residue (W180) of FAAP20 outside the compact UBZ module plays an indispensable role in ubiquitin recognition by FAAP20 in vitro and in efficient ICL DNA repair in vivo . ( A ) Measurement of binding affinity between WT FAAP20 and WT ubiquitin by ITC. ( B ) Mutation of W180A in FAAP20 abolished ubiquitin binding in ITC measurements. ( C ) U2OS cells stably expressing siRNA-resistant FAAP20 wild-type, C147A and C150A (CA), or W180A (WA) were transfected with siRNA against FAAP20 (siF20) for 48 h, and cell viability was determined 6 days after the treatment of indicated doses of mitomycin C. ( D ) Immunoblot analysis of U2OS cells in (C) harvested 72 h after siRNA treatment.

    Journal: Nucleic Acids Research

    Article Title: Ubiquitin recognition by FAAP20 expands the complex interface beyond the canonical UBZ domain

    doi: 10.1093/nar/gku1153

    Figure Lengend Snippet: The C-terminal tryptophan residue (W180) of FAAP20 outside the compact UBZ module plays an indispensable role in ubiquitin recognition by FAAP20 in vitro and in efficient ICL DNA repair in vivo . ( A ) Measurement of binding affinity between WT FAAP20 and WT ubiquitin by ITC. ( B ) Mutation of W180A in FAAP20 abolished ubiquitin binding in ITC measurements. ( C ) U2OS cells stably expressing siRNA-resistant FAAP20 wild-type, C147A and C150A (CA), or W180A (WA) were transfected with siRNA against FAAP20 (siF20) for 48 h, and cell viability was determined 6 days after the treatment of indicated doses of mitomycin C. ( D ) Immunoblot analysis of U2OS cells in (C) harvested 72 h after siRNA treatment.

    Article Snippet: Protein constructs and cloning The DNA sequence of residues 140–180 of human FAAP20 isoform 2 was synthesized; the polymerase chain reaction amplified DNA was double digested and ligated into a modified pET15b vector (EMD Biosciences, Inc.) between the NdeI and XhoI restriction sites.

    Techniques: In Vitro, In Vivo, Binding Assay, Mutagenesis, Stable Transfection, Expressing, Transfection

    W104 mutants discriminate between different SinR tetramer models. Ribbon representations are shown of the two proposed models of SinR tetramers, in which the DNA binding domains are colored pale green and the tetramerization domains are colored pale blue. Dashed lines represent the linkers between the domains that cannot be modeled in any SinR-containing structure because of flexibility. The N and C termini are labeled, where they are visible, and the side chain for W104 is drawn in “stick” format and colored and labeled in red. For the model in panel A (from Colledge et al. [ 12 ] PDB ID 2YAL ), the structure of the C-terminal domain of SinR was solved in isolation. Note that the DNA-binding domains of SinR in this model are too far apart to be consistent with binding to pairs of sin boxes as found in promoters of genes regulated by SinR, but the position of W104 in this model, critical to tetramerization, is consistent with the biochemistry and genetics presented here. For the model in panel B (from Newman et al. [ 13 ] PDB ID 3ZKC ), the structure of SinR bound to DNA is described; for clarity, the DNA is not included in this panel. Note that in the model in panel B, W104 plays no role in self-assembly of SinR.

    Journal: mBio

    Article Title: Selective Pressure for Biofilm Formation in Bacillus subtilis: Differential Effect of Mutations in the Master Regulator SinR on Bistability

    doi: 10.1128/mBio.01464-18

    Figure Lengend Snippet: W104 mutants discriminate between different SinR tetramer models. Ribbon representations are shown of the two proposed models of SinR tetramers, in which the DNA binding domains are colored pale green and the tetramerization domains are colored pale blue. Dashed lines represent the linkers between the domains that cannot be modeled in any SinR-containing structure because of flexibility. The N and C termini are labeled, where they are visible, and the side chain for W104 is drawn in “stick” format and colored and labeled in red. For the model in panel A (from Colledge et al. [ 12 ] PDB ID 2YAL ), the structure of the C-terminal domain of SinR was solved in isolation. Note that the DNA-binding domains of SinR in this model are too far apart to be consistent with binding to pairs of sin boxes as found in promoters of genes regulated by SinR, but the position of W104 in this model, critical to tetramerization, is consistent with the biochemistry and genetics presented here. For the model in panel B (from Newman et al. [ 13 ] PDB ID 3ZKC ), the structure of SinR bound to DNA is described; for clarity, the DNA is not included in this panel. Note that in the model in panel B, W104 plays no role in self-assembly of SinR.

    Article Snippet: The sinR alleles were amplified from chromosomal DNA of the respective mutants using primer pair G8/G10 ( ) and were cloned between the NdeI and EcoRI sites of expression vector pET24a (Novagen).

    Techniques: Binding Assay, Labeling, Isolation

    Binding of wild type and suppressor SinR variants to DNA. Fluorescence polarization was used to determine the dissociation constants ( K D ) of SinR variants binding to operator DNA. Fluorescently labeled DNA (10 nM concentration) was incubated with various concentrations of SinR proteins, and the fluorescence polarization was measured in triplicate. The polarization data were fitted to a 1:1 binding model in Sigma Plot software to determine K D values, which are reported in Table 1 .

    Journal: mBio

    Article Title: Selective Pressure for Biofilm Formation in Bacillus subtilis: Differential Effect of Mutations in the Master Regulator SinR on Bistability

    doi: 10.1128/mBio.01464-18

    Figure Lengend Snippet: Binding of wild type and suppressor SinR variants to DNA. Fluorescence polarization was used to determine the dissociation constants ( K D ) of SinR variants binding to operator DNA. Fluorescently labeled DNA (10 nM concentration) was incubated with various concentrations of SinR proteins, and the fluorescence polarization was measured in triplicate. The polarization data were fitted to a 1:1 binding model in Sigma Plot software to determine K D values, which are reported in Table 1 .

    Article Snippet: The sinR alleles were amplified from chromosomal DNA of the respective mutants using primer pair G8/G10 ( ) and were cloned between the NdeI and EcoRI sites of expression vector pET24a (Novagen).

    Techniques: Binding Assay, Fluorescence, Labeling, Concentration Assay, Incubation, Software

    Inhibition of SinR variants at different SinI concentrations. SinI was titrated against SinR variants prebound to the native operator site in order to analyze the ability of SinR proteins to bind to the antagonist SinI. Fluorescently labeled DNA (10 nM concentration) was preincubated with 5 µM of SinR, followed by a SinI titration and the measurement of fluorescence polarization. The polarization data at various SinI concentrations have been normalized to represent the percentages of DNA bound to SinR based on the measured levels of polarization of both free DNA and the DNA:SinR complex; for all of the SinR proteins, the DNA binding is saturated at 5 µM SinR and 10 nM DNA. The bars show the mean values and standard deviations of three measurements.

    Journal: mBio

    Article Title: Selective Pressure for Biofilm Formation in Bacillus subtilis: Differential Effect of Mutations in the Master Regulator SinR on Bistability

    doi: 10.1128/mBio.01464-18

    Figure Lengend Snippet: Inhibition of SinR variants at different SinI concentrations. SinI was titrated against SinR variants prebound to the native operator site in order to analyze the ability of SinR proteins to bind to the antagonist SinI. Fluorescently labeled DNA (10 nM concentration) was preincubated with 5 µM of SinR, followed by a SinI titration and the measurement of fluorescence polarization. The polarization data at various SinI concentrations have been normalized to represent the percentages of DNA bound to SinR based on the measured levels of polarization of both free DNA and the DNA:SinR complex; for all of the SinR proteins, the DNA binding is saturated at 5 µM SinR and 10 nM DNA. The bars show the mean values and standard deviations of three measurements.

    Article Snippet: The sinR alleles were amplified from chromosomal DNA of the respective mutants using primer pair G8/G10 ( ) and were cloned between the NdeI and EcoRI sites of expression vector pET24a (Novagen).

    Techniques: Inhibition, Labeling, Concentration Assay, Titration, Fluorescence, Binding Assay

    In vitro expression and enzymatic analysis of WspA protein. (a) Truncation of WspA1 for in vitro expression. (b) Induction of Wsp A and Wsp B in the E. coli BL21/pET28 expression system and their purification. P, total proteins in the absence of IPTG induction; IP, total proteins in the presence of IPTG induction; W1 and W2, washing fractions; E, elution fractions; D, dialyzed proteins; M, marker protein. (c) Blue-white test of transgenic E. coli cells on separate LB plates supplemented with X-Gal as the substrate. (d) In vitro activity analysis of purified Wsp A in X-Gal buffer (pH 7.5). (e) Comparative activity analysis in 0.1 M PBS buffer (pH 7.5) using ONPG as the substrate. Reactions were performed at 37°C. Data shown are means ± SD ( n = 4). BSA, 50 μg/ml; Wsp A , 10 μg/ml; Wsp B , 27 μg/ml. Boiling conditions, 100°C for 15 min.

    Journal: Applied and Environmental Microbiology

    Article Title: Flexibility-Rigidity Coordination of the Dense Exopolysaccharide Matrix in Terrestrial Cyanobacteria Acclimated to Periodic Desiccation

    doi: 10.1128/AEM.01619-17

    Figure Lengend Snippet: In vitro expression and enzymatic analysis of WspA protein. (a) Truncation of WspA1 for in vitro expression. (b) Induction of Wsp A and Wsp B in the E. coli BL21/pET28 expression system and their purification. P, total proteins in the absence of IPTG induction; IP, total proteins in the presence of IPTG induction; W1 and W2, washing fractions; E, elution fractions; D, dialyzed proteins; M, marker protein. (c) Blue-white test of transgenic E. coli cells on separate LB plates supplemented with X-Gal as the substrate. (d) In vitro activity analysis of purified Wsp A in X-Gal buffer (pH 7.5). (e) Comparative activity analysis in 0.1 M PBS buffer (pH 7.5) using ONPG as the substrate. Reactions were performed at 37°C. Data shown are means ± SD ( n = 4). BSA, 50 μg/ml; Wsp A , 10 μg/ml; Wsp B , 27 μg/ml. Boiling conditions, 100°C for 15 min.

    Article Snippet: The recombinant E. coli cells with an optical density at 600 nm (OD600 ) of 0.6 to 0.8 after culture with shaking at 200 rpm at 37°C were subjected to induction with 0.2 mM IPTG (isopropyl-β- d -thiogalactopyranoside) for 6 h. The cells were broken by ultrasonication for protein extraction, and the crude proteins were purified with His-Bind resin (Novagen, USA).

    Techniques: In Vitro, Expressing, Purification, Marker, Transgenic Assay, Activity Assay