Journal: Microbial Biotechnology

Article Title: Identification of the EH CRISPR‐Cas9 system on a metagenome and its application to genome engineering

doi: 10.1111/1751-7915.14266

Figure Lengend Snippet: Schematic representation of the EH CRISPR‐Cas9 locus and domains of the associated Cas9 protein. The EH CRISPR‐Cas9 locus comprises three cas genes in the order of cas9 (denoted ehcas9 ) – cas1 – cas2 (boxes pointing toward the direction of transcription), and two 36‐bp CRISPR units (Repeat, white rectangles) separated by a 29‐bp spacer (diamond). The location of a putative tracrRNA gene is represented as a black arrow pointing toward the predicted direction of transcription. The EHCas9 protein domains (depicted below the locus representation) inferred from the alignment with orthologues (Figure ), include the RuvC (I, II and III motifs), Bridge Helix (BH), recognition (REC), HNH, Phosphate Lock Loop (PLL), WED and PAM‐Interacting (PI) domains.

Article Snippet: For heterologous expression of EHCas9 protein, E . coli codon‐optimized ehcas9 gene supplied by NZYtech was fused to an N‐terminal hexahistidine tag under a lac /IPTG‐inducible promoter in a pHTP1 vector, generating pMML22 plasmid.

Techniques: CRISPR

Journal: Microbial Biotechnology

Article Title: Identification of the EH CRISPR‐Cas9 system on a metagenome and its application to genome engineering

doi: 10.1111/1751-7915.14266

Figure Lengend Snippet: Evolutionary relationship of EHCas9. Phylogenetic tree of EHCas9 and 798 orthologues. Clades II (dark green), III (purple), IV (orange) and V (dark blue) belong to subtype II‐A, clade I (pink) to subtype II‐B, and clades VI (yellow), VII (blue), VIII (green), IX (red) and X (lilac) to subtype II‐C. Cas9 of Sulfitobacter donghicola (SdoCas9) and orthologues commonly used for genome editing (SaCas9: Staphylococcus aureus Cas9; SpCas9: Streptococcus pyogenes Cas9; NmCas9: Neisseria meningitidis Cas9; CjCas9: Campylobacter jejuni Cas9; CdCas9: Corynebacterium diphteriae Cas9; StCas9: Streptococcus thermophilus Cas9) are labelled at their approximate position in the tree.

Article Snippet: For heterologous expression of EHCas9 protein, E . coli codon‐optimized ehcas9 gene supplied by NZYtech was fused to an N‐terminal hexahistidine tag under a lac /IPTG‐inducible promoter in a pHTP1 vector, generating pMML22 plasmid.

Techniques:

Journal: Microbial Biotechnology

Article Title: Identification of the EH CRISPR‐Cas9 system on a metagenome and its application to genome engineering

doi: 10.1111/1751-7915.14266

Figure Lengend Snippet: EHCas9 PAM screening and validation. Plasmids containing a spectinomycin resistance gene (SpcR) and distinct sequences in the PAM region (green) next to a target (red) matching the spacer (red diamond) of a CRISPR array (Array) composed of two repeats (blue rectangles; experiments in E . coli ) or the spacer region of a sgRNA (in vitro screening) were subjected to cleavage by EHCas9. (A) Schematic representation of the PAM screening in E . coli cells. E . coli cells harbouring pMML03 (tracrRNA carrier) and either the pMML02 (EHCas9‐expressing) or the pMML01 (EH‐Cas9‐devoid) plasmid were transformed with a 3‐nt (from the 2nd to the 4th position) PAM library. Plasmids pMML01 and pMML02 encode a constitutively expressed CRISPR array. Transformants were selected on spectinomycin plates, and the PAM region was sequenced by High Throughput Sequencing of PCR products of the plasmids purified from pooled colonies (PAM depletion analysis). A logo (PAM logo) of the sequences in the PAM region preferred by EHCas9 for target cleavage was generated from the PAM fold‐change calculated after comparing PAM frequencies in pMML02 carriers with the corresponding values from cells carrying pMML01. Nucleotide positions 3′ from the end of the spacer‐matching strand of the target are indicated in the PAM logo. (B) Schematic representation of the in vitro PAM screening. EHCas9 protein was preincubated with EH sgRNA to generate EHCas9:sgRNA nucleoprotein complexes and mixed (cleavage reaction) with a plasmid library (7‐nt PAM library) with randomized nucleotides from the 1st to the 7th position of the PAM region. Adapters were ligated to the linearized DNA fragments and a logo (PAM logo) of the nucleotide sequences in the randomized region preferred by EHCas9 for target cleavage was generated from the frequencies of each sequence calculated after llumina sequencing of PAM‐sided PCR products. Nucleotide positions 3′ from the end of the spacer‐matching strand of the target are indicated in the PAM logo. (C) Schematic representation of the in vivo PAM validation in E . coli . E . coli cells bearing pMML03 and either pMML02 or pMML01 were transformed with a plasmid containing a target flanked by 5′‐TACCG‐3′ (PAM‐less plasmid), 5′‐TGGAG‐3′ (TGGAG plasmid), 5′‐TGGCG3′ (TGGCG plasmid), 5′‐TGGGG‐3′ (TGGGG plasmid) or 5′‐TGGTG‐3′ (TGGTG plasmid) in the PAM region. Transformants were selected on spectinomycin plates and the efficiency of transformation (number of colony‐forming units – CFU – per mg of plasmid DNA) of E . coli cells carrying pMML02 (+ EHCas9) or pMML01 (− EHCas9) was represented. Data are the mean of three biological replicates (error bars correspond to the standard deviation).

Article Snippet: For heterologous expression of EHCas9 protein, E . coli codon‐optimized ehcas9 gene supplied by NZYtech was fused to an N‐terminal hexahistidine tag under a lac /IPTG‐inducible promoter in a pHTP1 vector, generating pMML22 plasmid.

Techniques: Biomarker Discovery, CRISPR, In Vitro, Expressing, Plasmid Preparation, Transformation Assay, Next-Generation Sequencing, Purification, Generated, Sequencing, In Vivo, Standard Deviation

Journal: Microbial Biotechnology

Article Title: Identification of the EH CRISPR‐Cas9 system on a metagenome and its application to genome engineering

doi: 10.1111/1751-7915.14266

Figure Lengend Snippet: Representative agarose gel electrophoreses of in vitro EHCas9‐mediated DNA cleavage reactions. Except indicated otherwise, experiments were performed under the following standard conditions: 30 min reaction time at 37°C, 20 mM MgCl 2 , 25 nM of an 840 bp linear DNA containing a target sequence and a compatible PAM, preincubated (15 min at 37°C) EHCas9:EH sgRNA mixtures (0.5 mM EHCas9 and 0.5 mM EH sgRNA concentration in the digestion reaction). The length of relevant bands (in kb) of a linear dsDNA molecular weight marker (M) and the position of bands corresponding to expected cut and uncut DNA substrates are indicated. (A) Samples from standard digestion reactions, mixed either after protein:guide preincubation (lane 2) or without preincubation (lane 7), and from reactions with a missing component (MgCl 2 , lane 3; a compatible PAM next to the target, lane 4; a guide, lane 5; the protein, lane 6) are included. (B) Samples from digestion reactions under standard conditions but for the protein concentration (up to 500 nM). (C) Samples from standard reactions incubated for up to 40 min. (D) Samples of digestions carried out under standard conditions except by the incubation temperature (from 20°C to 45°C).

Article Snippet: For heterologous expression of EHCas9 protein, E . coli codon‐optimized ehcas9 gene supplied by NZYtech was fused to an N‐terminal hexahistidine tag under a lac /IPTG‐inducible promoter in a pHTP1 vector, generating pMML22 plasmid.

Techniques: Agarose Gel Electrophoresis, In Vitro, Sequencing, Concentration Assay, Molecular Weight, Marker, Protein Concentration, Incubation

Journal: Microbial Biotechnology

Article Title: Identification of the EH CRISPR‐Cas9 system on a metagenome and its application to genome engineering

doi: 10.1111/1751-7915.14266

Figure Lengend Snippet: Prokaryotic genome editing with EHCas9. (A) Overview of the single‐step general procedure for positive selection of genome‐edited E . coli . E . coli cells harbouring a plasmid (e.g., pKD46; ampicillin resistance, Amp R ) encoding Lambda Red recombination proteins (Gam, Beta, Exo) are co‐transformed with an antibiotic resistance (Ab R ) selectable plasmid carrying inducible ehcas9 gene and a sgRNA targeting a sequence within the gene of interest (GOI) and a linear dsDNA template matching both sides of the target (flanking sites, FS). Recombination between the template and the flanking sites in the gene mediated by the Lambda Red recombination machinery (RM) will result in the deletion of the target sequence. The Cas9:sgRNA ribonucleoprotein complex will produce double‐strand breaks in the non‐edited target, often leading to cell death. Colonies grown at 37°C expressing Cas9 are selected on plates (i.e., arabinose and antibiotic‐containing medium) and screened through PCR amplification and agarose gel electrophoresis to confirm the deletion. (B) An agarose gel electrophoresis of PCR products from the region surrounding pyrF (the gene of interest) in E . coli cells expressing the Lambda Red system from pKD46. DNA used for amplification was purified from chloramphenicol‐resistant colonies grown in the presence of arabinose after co‐transformation with a recombination template matching the flanks of pyrF (recombination would lead to a ca. 0.6 kb deletion), and a pBAD33‐derivative plasmid (providing chloramphenicol resistance) encoding either both EHCas9 and an EH sgRNA that targets the pyrF gene (+EHCas9) or only the EH sgRNA (−EHCas9). Each lane corresponds to a transformant clone. Bands running as linear DNA fragments with the length of the original (ca. 1 kb; WT) and the recombinant (ca. 0.5 kb; Mutant) pyrF region are indicated. The length of relevant bands of a linear dsDNA molecular weight marker is indicated.

Article Snippet: For heterologous expression of EHCas9 protein, E . coli codon‐optimized ehcas9 gene supplied by NZYtech was fused to an N‐terminal hexahistidine tag under a lac /IPTG‐inducible promoter in a pHTP1 vector, generating pMML22 plasmid.

Techniques: Selection, Plasmid Preparation, Transformation Assay, Sequencing, Expressing, Amplification, Agarose Gel Electrophoresis, Purification, Recombinant, Mutagenesis, Molecular Weight, Marker

Journal: Microbial Biotechnology

Article Title: Identification of the EH CRISPR‐Cas9 system on a metagenome and its application to genome engineering

doi: 10.1111/1751-7915.14266

Figure Lengend Snippet: EHCas9‐mediated genome editing in mouse cells. (A) Workflow of the Cas9 genome editing assay performed in mouse N2a cell cultures. A plasmid harbouring the cas9 gene ( ehcas9 or spcas9 ) fused to a nuclear localization signal (NLS) was co‐transfected into N2a cells with another plasmid carrying the corresponding sgRNA‐encoding sequence under constitutive promoters (CMV and U6, respectively). Insertions and deletions (INDELs) in the guide complementary region were analysed by HTS of PCR amplicons from the region encompassing the target site. (B) Percentage of reads with INDELs ( n = 3 each, mean ± SD) obtained for 4 target regions ( Oca2 .2, Oca2 .3, Oca2 .4, Lrmda .1) after cell transfection with plasmids carrying SpCas9 and Sp sgRNA (SpCas9.sgRNA; blue), EHCas9 and EH sgRNA (EHCas9.sgRNA; yellow), or EHCas9 (EHCas9; green). Data from non‐transfected cells (N2a; orange) are also included. The PAM sequence of each target site is indicated. (C) Alignment of the 10 most abundant alleles revealed for Oca2 .3 in EHCas9:EH sgRNA experiments. Allele frequency (mean of three biological replicates) is displayed. Left column denotes deletion positions referred to the expected cleavage‐site (dashed line) (e.g., −2:1D, one nucleotide deletion at position −2 from the cleavage site). Top line, original target region. The target sequence is coloured in orange and the PAM region in blue.

Article Snippet: For heterologous expression of EHCas9 protein, E . coli codon‐optimized ehcas9 gene supplied by NZYtech was fused to an N‐terminal hexahistidine tag under a lac /IPTG‐inducible promoter in a pHTP1 vector, generating pMML22 plasmid.

Techniques: Plasmid Preparation, Transfection, Sequencing

Journal: Journal of Biological Engineering

Article Title: Directed evolution of bright mutants of an oxygen-independent flavin-binding fluorescent protein from Pseudomonas putida

doi: 10.1186/1754-1611-6-20

Figure Lengend Snippet: Sequence alignment between FbFP and YTVA ( B. subtilis ). Sequence alignment reveals close agreement between FbFP from P. putida and YTVA from B. subtilis , with 37% identical and 62% similar amino acids shared between the two proteins. In this way, the YTVA structure serves as an ideal template for molecular modeling of the FbFP. Amino acids selected for site saturation mutagenesis are indicated in red and underlined.

Article Snippet: The Pseudomonas putida KT2440 gene coding for the FbFP [RefSeq ID: NP_744883.1] was synthesized by GenScript (Piscataway, NJ).

Techniques: Sequencing, Mutagenesis

Journal: Journal of Bacteriology

Article Title: Allosteric Regulation of Lactobacillus plantarum Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase (Xfp)

doi: 10.1128/JB.02380-14

Figure Lengend Snippet: Effect of pH on L. plantarum Xfp and C. neoformans Xfp2 activity. Enzyme reactions were performed using F6P and Pi K0.5 concentrations for L. plantarum Xfp and C. neoformans Xfp2. Activity begins to decrease at pH values above 6.0 for C. neoformans Xfp2 and above 6.5 for L. plantarum Xfp.

Article Snippet: The recombinant plasmid pET21b- XFP2 synthesized by GenScript was transformed into the E. coli expression strain RosettaBlue (Novagen).

Techniques: Activity Assay

Journal: Journal of Bacteriology

Article Title: Allosteric Regulation of Lactobacillus plantarum Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase (Xfp)

doi: 10.1128/JB.02380-14

Figure Lengend Snippet: Apparent kinetic parameters for L. plantarum Xfp and C. neoformans Xfp2

Article Snippet: The recombinant plasmid pET21b- XFP2 synthesized by GenScript was transformed into the E. coli expression strain RosettaBlue (Novagen).

Techniques:

Journal: Journal of Bacteriology

Article Title: Allosteric Regulation of Lactobacillus plantarum Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase (Xfp)

doi: 10.1128/JB.02380-14

Figure Lengend Snippet: Effect of nonphosphorylated PEP analogs on L. plantarum Xfp and C. neoformans Xfp2 activities. Reactions were performed using L. plantarum Xfp (A) and C. neoformans Xfp2 (B) F6P and Pi K0.5 substrate concentrations. Concentrations of 8 mM (dark gray) and 16 mM (light gray) for each nonphosphorylated PEP analog were tested in triplicate. Activity is reported as a percentage of activity with no ligand present.

Article Snippet: The recombinant plasmid pET21b- XFP2 synthesized by GenScript was transformed into the E. coli expression strain RosettaBlue (Novagen).

Techniques: Activity Assay

Journal: Journal of Bacteriology

Article Title: Allosteric Regulation of Lactobacillus plantarum Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase (Xfp)

doi: 10.1128/JB.02380-14

Figure Lengend Snippet: Half-maximal inhibitory concentrations (IC 50 s)

Article Snippet: The recombinant plasmid pET21b- XFP2 synthesized by GenScript was transformed into the E. coli expression strain RosettaBlue (Novagen).

Techniques:

Journal: Plant Communications

Article Title: Structural Insights into the Catalytic Mechanism of a Plant Diterpene Glycosyltransferase SrUGT76G1

doi: 10.1016/j.xplc.2019.100004

Figure Lengend Snippet: A Network of SG Biosynthesis. Nine SrUGT76G1-catalyzing reactions are indicated by red arrows. The bold arrows indicate a major in planta route to form rebaudioside M (RebM). The diterpenoid aglycones are indicated in gray. The structure of steviol is shown on the upper right. Blue circles represent C13- O -linked glucosyl (Glc1), yellow circles represent C19- O -linked glucosyl (Glc1′), green circles represent 1,2- O -linked glucosyl (Glc2/Glc2′), and red circles represent 1,3- O -linked glucosyl (Glc3/Glc3′). Biochemical validation of SrUGT76G1-catalyzing reactions is shown in Supplemental Figure 2 .

Article Snippet: The native SrUGT76G1 gene (codon-optimized in E . coli ; Genscript, Nanjing) was subcloned from SrUGT76G1 -cloning vector by PCR with specific primer pairs , digested with Bam HI and Hin dIII, and ligated into Bam HI/ Hin dIII-predigested vector pETDuet-1, resulting in plasmid pQZ11.

Techniques: Biomarker Discovery

Journal: Plant Communications

Article Title: Structural Insights into the Catalytic Mechanism of a Plant Diterpene Glycosyltransferase SrUGT76G1

doi: 10.1016/j.xplc.2019.100004

Figure Lengend Snippet: Overall Structure of SrUGT76G1. (A–D) SrUGT76G1 complex with UDP (A) , UDP-xylose (B) , UDP and steviolmonoside (Sm) (C) , UDP and rebaudioside B (RebB) (D) . (E) Three domains of the SrUGT76G1. NTD, N-terminal acceptor-binding domain; CTD, C-terminal donor-binding domain; LR, linker region.

Article Snippet: The native SrUGT76G1 gene (codon-optimized in E . coli ; Genscript, Nanjing) was subcloned from SrUGT76G1 -cloning vector by PCR with specific primer pairs , digested with Bam HI and Hin dIII, and ligated into Bam HI/ Hin dIII-predigested vector pETDuet-1, resulting in plasmid pQZ11.

Techniques: Binding Assay

Journal: Plant Communications

Article Title: Structural Insights into the Catalytic Mechanism of a Plant Diterpene Glycosyltransferase SrUGT76G1

doi: 10.1016/j.xplc.2019.100004

Figure Lengend Snippet: Snapshot of Differing Ligand-Binding Status and Conformation Change. SrUGT76G1 structures accommodating different substrates were superposed. (A) Superposition of UDP-SrUGT76G1 and UDPX-SrUGT76G1 revealed the location of activated sugar; (B) superposed UDPX-SrUGT76G1 and UDP-Sm-SrUGT76G1 represent a state when sugar acceptor is coming close to sugar donor; (C) superposed UDPX-SrUGT76G1 and UDP-RebB-SrUGT76G1 represent a state when glucosylated product is formed; (D) superposition of UDP-RebB-SrUGT76G1 and UDP-Sm-SrUGT76G1 revealed the location of C2′-linked glucose; (E) two disordered regions (black spot circles) in UDP-SrUGT76G1 complex structures; (F) binding of sugar acceptor has a major effect on the conformational change of NTD. Magenta, UDP-SrUGT76G1; orange, UDPX-SrUGT76G1; green, UDP-Sm-SrUGT76G1; blue, UDP-RebB-SrUGT76G1.

Article Snippet: The native SrUGT76G1 gene (codon-optimized in E . coli ; Genscript, Nanjing) was subcloned from SrUGT76G1 -cloning vector by PCR with specific primer pairs , digested with Bam HI and Hin dIII, and ligated into Bam HI/ Hin dIII-predigested vector pETDuet-1, resulting in plasmid pQZ11.

Techniques: Ligand Binding Assay, Binding Assay

Journal: Plant Communications

Article Title: Structural Insights into the Catalytic Mechanism of a Plant Diterpene Glycosyltransferase SrUGT76G1

doi: 10.1016/j.xplc.2019.100004

Figure Lengend Snippet: SrUGT76G1 Possess a Larger Acceptor-Binding Pocket. Substrate-binding pockets for comparison are: (A) UDP-RebB-SrUGT76G1; (B) VvGT1 (PDB: 2C1Z ); (C) Os79 (PDB: 5TMD ); (D) UGT72B1 (PDB: 2VCE ). Although the sugar donor binding pockets are in uniform size, the sugar-acceptor binding pocket in SrUGT76G1 is obviously larger than those accommodating flavonoid, sesquiterpene, and small aromatic compounds.

Article Snippet: The native SrUGT76G1 gene (codon-optimized in E . coli ; Genscript, Nanjing) was subcloned from SrUGT76G1 -cloning vector by PCR with specific primer pairs , digested with Bam HI and Hin dIII, and ligated into Bam HI/ Hin dIII-predigested vector pETDuet-1, resulting in plasmid pQZ11.

Techniques: Binding Assay, Comparison

Journal: Plant Communications

Article Title: Structural Insights into the Catalytic Mechanism of a Plant Diterpene Glycosyltransferase SrUGT76G1

doi: 10.1016/j.xplc.2019.100004

Figure Lengend Snippet: Molecular Basis for Diterpene Skeleton Recognition. (A–E) Hydrophobic pocket observed in the complex of UDP-RebB-SrUGT76G1 (A) . Key residues involved in diterpene aglycone recognition (B). Mutations of hydrophobic residues on α2 helix and α8 helix altered substrate preference (C) . Changes of in vitro enzymatic activity are calculated as log 2 (relative conversion rate); the activity of wild-type SrUGT76G1 was set as 1 (100%). Classification of substrate types: (I) substrates with C19- O -glucosyl; (II) substrates with C13- O -glucosyl; (III) substrates with C13/C19- O -sophorosyl. (D) Simulation of Phe mutation in active sites restrains the size of binding pocket. (E) Mutants G87F, I199F, L204F show enhanced activity toward flavonoid glycoside isoorientin ( 1 ). ST, isoorientin standard; WT, wild-type SrUGT76G1. (F) Mutation of G87 and L204 to Phe impair the glycosylation efficiency of diterpenoid substrates (i.e., steviolmonoside and steviolbioside).

Article Snippet: The native SrUGT76G1 gene (codon-optimized in E . coli ; Genscript, Nanjing) was subcloned from SrUGT76G1 -cloning vector by PCR with specific primer pairs , digested with Bam HI and Hin dIII, and ligated into Bam HI/ Hin dIII-predigested vector pETDuet-1, resulting in plasmid pQZ11.

Techniques: In Vitro, Activity Assay, Mutagenesis, Binding Assay, Glycoproteomics

Journal: Plant Communications

Article Title: Structural Insights into the Catalytic Mechanism of a Plant Diterpene Glycosyltransferase SrUGT76G1

doi: 10.1016/j.xplc.2019.100004

Figure Lengend Snippet: Binding of Sugar Moieties Involves S147, H155, and T284. (A) Sugar-binding pocket observed in the complex of UDP-RebB-SrUGT76G1. (B–F) Key residues interacting with C13-linked sugar (Glc1), C2′-linked sugar (Glc2), and C3′-linked sugar (Glc3) in SrUGT76G1 (B) ; in vitro enzymatic activities of H155 mutants (C) ; in vitro enzymatic activities of S147 mutants (D) ; in vitro enzymatic activities of T284 mutants toward RebA and RebD (E) ; manufacture of RebM using SrUGT76G1 T284S mutant (F) . ST M1, a standard mixture of RebI and RebA; ST M2, a standard mixture of RebA, RebD, and RebM; 651, fermentation extract of E . coli BL21(DE3) contains plasmid pHJ651; 830, fermentation extract of E . coli BL21(DE3) contains plasmid pHJ830. For detailed strain information, see .

Article Snippet: The native SrUGT76G1 gene (codon-optimized in E . coli ; Genscript, Nanjing) was subcloned from SrUGT76G1 -cloning vector by PCR with specific primer pairs , digested with Bam HI and Hin dIII, and ligated into Bam HI/ Hin dIII-predigested vector pETDuet-1, resulting in plasmid pQZ11.

Techniques: Binding Assay, In Vitro, Mutagenesis, Plasmid Preparation

Journal: Molecular Microbiology

Article Title: Mutants of phage bIL67 RuvC with enhanced Holliday junction binding selectivity and resolution symmetry

doi: 10.1111/mmi.12343

Figure Lengend Snippet: Comparison of phage and cellular RuvC resolving enzymes. A. Alignment of selected phage RuvC proteins. Structural elements of phage bIL67, Escherichia coli ( Eco ) and Thermus thermophilus ( Tth ) proteins are indicated above and below the aligned sequences (pink bars, α-helix; cyan arrows, β-sheet). Conserved residues are highlighted in red (acidic; active site), blue (basic), cyan (others) and orange (F69 in Ec RuvC and F73 in Tth RuvC). Every ten residues are labelled as filled grey circles above the bIL67 RuvC sequence. Filled magenta circles indicate the location of bIL67 RuvC S10A, T11A, K40A, R46A, S109A, K110A, K120A, R121A, R124A, K125A and R124A+K125A substitution mutants. Sequence conservation in E. coli and T. thermophilus RuvC is taken from the Pfam database entry, PF02075, highlighting the most highly conserved residues among bacterial RuvC family proteins. Conserved residues in phage RuvC are based on 24 orthologues using a similar approach. The percentage identity refers to homology of each protein with bIL67 RuvC. Green arrows indicate the positions of insertions to generate N- and C-terminal 67- Ec RuvC and Ec -67RuvC hybrids (see ). Accession numbers of selected lactococcal phage RuvC proteins from bIL67 (NP_042322), bIL66 (AAA99046), Q54 (YP_762587) and Streptococcus pyogenes inducible phage EJ-1 (NP_945263). B. Structural superimposition based upon their central β-sheets of monomers of 67RuvC (cyan) and Ec RuvC (dark magenta) shown in cartoon representation as in but with α-helices shown as cylinders. Helices and termini are labelled. C. Superimpostion of the catalytically critical residues at the active sites of 67RuvC (cyan), Ec RuvC (dark magenta) and Tth RuvC (olive green). Side-chains are shown in stick representation with the rest of the structure in cartoon form. The 67RuvC residue labelled D8 is an asparagine in the crystal structure shown. The Mg 2+ cation (green sphere) and solvent ligands (red spheres) from the 67RuvC structure are shown for reference. D. Alignment of dimers of 67RuvC and Ec RuvC based upon superimposition of the αB dimer interface helices. Structures are represented and coloured as in (B).

Article Snippet: An E. coli codon-optimized version ( ) of the Lactococcus lactis phage bIL67 ruvC ( ORF23 ) gene (Schouler et al ., ) was synthesized and inserted into the T7 expression vector pET24a(+) at NdeI and BamHI sites by GenScript to create p67RuvCwt.

Techniques: Comparison, Sequencing, Residue, Solvent

Journal: Protein Science : A Publication of the Protein Society

Article Title: Single molecule force spectroscopy reveals the effect of BiP chaperone on protein folding

doi: 10.1002/pro.3137

Figure Lengend Snippet: Experimental optical tweezer setup and the effect of BiP binding on MJ0336 folding and unfolding. A: The substrate protein MJ0336 is tethered between two dsDNA handles through disulfide bonds at its N and C terminus. These handles are modified at the 5′ ends with digoxigenin and biotin to bind the respective polystyrene beads coated with anti‐digoxigenin and streptavidin, respectively. The anti‐digoxigenin bead is held in an optical trap and the streptavidin bead is attached to a micropipette by suction. B: At the left, force‐extension curves showing the consecutive pulling cycles of MJ0336 at a constant speed of 100 nm/s in the absence of BiP. At the right, a schematic representation of the mechanical unfolding and refolding of MJ0366, with the respective unfolding and refolding events. C: At the left, force‐extension curves of MJ0366 in the presence of BiP. At the right, a schematic representation of the mechanical unfolding and inhibition of refolding of MJ0366 due to the binding of BiP (orange) to the unfolded state, with their respective unfolding and lost refolding event. Pulling traces where unfolding occurs at high forces are shown in red and relaxation traces with refolding events at lower forces are shown in blue. In the upper image (B), the force spectroscopy experiments without BiP and with 2 mM ATP show that the pulling of the protein leads to its unfolding and later refolding in all cycles. In the lower image (C), experiments were performed with 1 µM BiP and 2 mM ATP, and unfolding and refolding events were lost (second and third force‐extension curves), and regained after some time (fourth force‐extension curve).

Article Snippet: MJ0366 construct preparation An E. coli codon optimized synthetic gene was synthesized corresponding to the coding sequence of MJ0366 (GenScript, USA) and was subcloned into a modified pET‐21b vector between NcoI and EcoRI sites, resulting in a fusion with the MJ0366 reading frame, followed by a TEV protease cleavage site and His‐tag at the C‐terminus.

Techniques: Binding Assay, Modification, Inhibition, Force Spectroscopy