Journal: Engineering in Life Sciences

Article Title: Effects of heavy‐ion beam irradiation on avermectin B1a and its analogues production by Streptomyces avermitilis

doi: 10.1002/elsc.201800094

Figure Lengend Snippet: Comparison of the production of avermectin B1a and its analogs by fermentation of wild‐type and mutants of ZJAV‐Y‐147 and ZJAV‐Y‐HS at 28°C in a water bath shaker for 240 h at 250 r/min

Article Snippet: In the same way, the production of an industrial overproducer increased from 3582 to 4450 mg/L and improved by 24%. table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Production of time (h), avermectin B1a and its analogs (μg/mL) S. avermitilis Avermectin 48 h 72 h 96 h 120 h 144 h 168 h 192 h 216 h 240 h ATCC 31267 (Wild‐type) B2b 178.64 111.63 25.71 40.51 30.64 48.39 55.45 67.97 105.23 B2a – 89.43 462.32 675.12 581.52 814.49 567.46 1401.23 1026.67 A2b 82.24 90.62 – – – – 24.67 64.27 145.92 A2a 106.19 315.94 630.7 771.45 407.23 842.39 1115.24 1267.36 1326.76 B1b – 56.53 119.62 190.73 153.05 197.08 186.91 243.89 391.93 B1a 42.12 180.93 346.30 521.64 730.92 965.83 1312.53 1827.6 2335.6 ZJAV‐Y‐147 (Mutant) A1a 88.96 79.33 190.24 239.23 233.52 318.65 404.43 511.52 711.37 B2b 179.56 91.56 – – – – 50.84 69.49 100.83 B2a – 95.17 407.63 423.42 476.25 563.09 946.28 1204.25 1726.23 A2b 30.68 93.62 – – – 19.72 52.08 119.92 152.56 A2a 56.78 323.24 490.73 662.47 575.43 681.14 1134.76 1290.72 1810.62 B1b – – 95.59 124.83 141.89 160.36 242.16 295.36 390.73 B1a 63.91 281.12 798.14 1621.23 2465.62 2676.43 3434.85 3835.24 4822.23 ZJAV‐Y‐HS (Mutant) A1a – 79.33 186.93 205.79 231.65 286.21 429.28 541.39 733.26 B2b 162.83 233.99 – – – – 78.22 96.76 121.37 B2a – 186.37 516.34 806.13 1109.33 114.45 1451.16 1677.26 1901.66 A2b 28.63 – – – – – 157.49 166.99 167.45 A2a 52.06 349.49 621.27 1258.34 1367.34 1348.24 1743.66 1801.84 1994.85 B1b – – 121.76 236.24 331.74 317.39 371.49 460.67 428.22 B1a 48.88 313.79 836.16 1558.89 2194.86 2487.87 3123.41 3694.18 4632.17 A1a – 110.22 236.63 393.34 547.89 566.47 658.87 755.74 826.73 Open in a separate window Comparison of the production of avermectin B1a and its analogs by fermentation of wild‐type and mutants of ZJAV‐Y‐147 and ZJAV‐Y‐HS at 28°C in a water bath shaker for 240 h at 250 r/min Among other analogue parameters studied here, the results demonstrated that the mutants ZJAV‐Y‐147 and ZJAV‐Y‐HS produced maximal levels of avermectin B1a after 240 h of fermentation in a medium of pH 7.5 using 5% (v/v) inoculum medium.

Techniques: Mutagenesis

Journal: Engineering in Life Sciences

Article Title: Effects of heavy‐ion beam irradiation on avermectin B1a and its analogues production by Streptomyces avermitilis

doi: 10.1002/elsc.201800094

Figure Lengend Snippet: Values of AVMs B1a and Total AVMS for wild‐type and mutants of ZJAV‐Y‐147 and ZJAV‐Y‐HS mutants for different subcultures ( n = 5)

Article Snippet: In the same way, the production of an industrial overproducer increased from 3582 to 4450 mg/L and improved by 24%. table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Production of time (h), avermectin B1a and its analogs (μg/mL) S. avermitilis Avermectin 48 h 72 h 96 h 120 h 144 h 168 h 192 h 216 h 240 h ATCC 31267 (Wild‐type) B2b 178.64 111.63 25.71 40.51 30.64 48.39 55.45 67.97 105.23 B2a – 89.43 462.32 675.12 581.52 814.49 567.46 1401.23 1026.67 A2b 82.24 90.62 – – – – 24.67 64.27 145.92 A2a 106.19 315.94 630.7 771.45 407.23 842.39 1115.24 1267.36 1326.76 B1b – 56.53 119.62 190.73 153.05 197.08 186.91 243.89 391.93 B1a 42.12 180.93 346.30 521.64 730.92 965.83 1312.53 1827.6 2335.6 ZJAV‐Y‐147 (Mutant) A1a 88.96 79.33 190.24 239.23 233.52 318.65 404.43 511.52 711.37 B2b 179.56 91.56 – – – – 50.84 69.49 100.83 B2a – 95.17 407.63 423.42 476.25 563.09 946.28 1204.25 1726.23 A2b 30.68 93.62 – – – 19.72 52.08 119.92 152.56 A2a 56.78 323.24 490.73 662.47 575.43 681.14 1134.76 1290.72 1810.62 B1b – – 95.59 124.83 141.89 160.36 242.16 295.36 390.73 B1a 63.91 281.12 798.14 1621.23 2465.62 2676.43 3434.85 3835.24 4822.23 ZJAV‐Y‐HS (Mutant) A1a – 79.33 186.93 205.79 231.65 286.21 429.28 541.39 733.26 B2b 162.83 233.99 – – – – 78.22 96.76 121.37 B2a – 186.37 516.34 806.13 1109.33 114.45 1451.16 1677.26 1901.66 A2b 28.63 – – – – – 157.49 166.99 167.45 A2a 52.06 349.49 621.27 1258.34 1367.34 1348.24 1743.66 1801.84 1994.85 B1b – – 121.76 236.24 331.74 317.39 371.49 460.67 428.22 B1a 48.88 313.79 836.16 1558.89 2194.86 2487.87 3123.41 3694.18 4632.17 A1a – 110.22 236.63 393.34 547.89 566.47 658.87 755.74 826.73 Open in a separate window Comparison of the production of avermectin B1a and its analogs by fermentation of wild‐type and mutants of ZJAV‐Y‐147 and ZJAV‐Y‐HS at 28°C in a water bath shaker for 240 h at 250 r/min Among other analogue parameters studied here, the results demonstrated that the mutants ZJAV‐Y‐147 and ZJAV‐Y‐HS produced maximal levels of avermectin B1a after 240 h of fermentation in a medium of pH 7.5 using 5% (v/v) inoculum medium.

Techniques:

Journal: Biochemistry

Article Title: Divergence in ubiquitin interaction and catalysis among the ubiquitin-specific protease family DUBs

doi: 10.1021/acs.biochem.6b00033

Figure Lengend Snippet: Steady-state kinetic analysis of USP2CD and USP21CD with double mutant ubiquitin.

Article Snippet: We also would like to thank Dr. Cheryl Arrowsmith for the USP21 expression vector deposited in Addgene (3MTN).

Techniques: Mutagenesis, Ubiquitin Proteomics

Journal: Biochemistry

Article Title: Divergence in ubiquitin interaction and catalysis among the ubiquitin-specific protease family DUBs

doi: 10.1021/acs.biochem.6b00033

Figure Lengend Snippet: The different interactions of Ub Lys48 side chain with USPs as revealed by the USP-Ub complex structures. Surface structures are represented by their electrostatic potential represented as red (negatively charged), white (neutral/hydrophobic), and blue (positively charged). (A) USP7 (PDB 1NBF) in complex with Ub (cyan). The zoomed-in view shows the pocket that Ub Lys48 binds to. (B) Ub Lys48 forms a bidentate interaction with USP7 Asp305 and Glu308 (orange). (C) USP2-Ub complex structures reveal two conformations of Ub Lys48. (D) Ub Lys48 (magenta) in USP2-Ub complex structure (PDB 3NHE) forms a hydrogen bond with USP2 Asp367. However, Ub Lys48 (cyan) in the USP2-Ub complex structure (PDB 2HD5) forms no hydrogen bond with USP2 residue. (E) USP21 (PDB 3I3T) in complex with ubiquitin (cyan). (F) USP21 Glu311 (yellow), which is structurally aligned to USP7 Asp305 and USP2 Asp367, does not form a hydrogen bond with Ub Lys48.

Article Snippet: We also would like to thank Dr. Cheryl Arrowsmith for the USP21 expression vector deposited in Addgene (3MTN).

Techniques: Residue, Ubiquitin Proteomics

Journal: Biochemistry

Article Title: Divergence in ubiquitin interaction and catalysis among the ubiquitin-specific protease family DUBs

doi: 10.1021/acs.biochem.6b00033

Figure Lengend Snippet: Interactions of Ub Leu73 and Lys6 with USPs. Ub is colored cyan. USP surface is colored according to the type of atoms: carbon (green), oxygen (red), nitrogen (blue) and sulfur (yellow). Ub Leu73 binds to a pocket in USP2 (A), USP7 (B), and USP21 (C). Ub Lys6 interacts with USP2 (D), USP7 (E), and USP21 (F) with no pocket identified.

Article Snippet: We also would like to thank Dr. Cheryl Arrowsmith for the USP21 expression vector deposited in Addgene (3MTN).

Techniques:

Journal: Biochemistry

Article Title: Divergence in ubiquitin interaction and catalysis among the ubiquitin-specific protease family DUBs

doi: 10.1021/acs.biochem.6b00033

Figure Lengend Snippet: Interaction of Ub Arg72 with USPs. (A) Ub Arg72 forms a hydrogen bond with a conserved glutamate in USP2 (PDB 2HD5, green), USP7 (PDB 1NBF, orange), USP8 (PDB 2GFO, magenta), and USP21 (PDB 3I3T, yellow) complex structures. (B) Binding of Ub Arg72 to USP7 (apo shown in grey, PDB 1NB8) may promote conformational changes that reposition the active site Cys223 to a catalytically competent conformation (orange).

Article Snippet: We also would like to thank Dr. Cheryl Arrowsmith for the USP21 expression vector deposited in Addgene (3MTN).

Techniques: Binding Assay

Journal: Nature Structural & Molecular Biology

Article Title: Structural basis of the specificity of USP18 toward ISG15

doi: 10.1038/nsmb.3371

Figure Lengend Snippet: ( a ) Immunoblot of lysates from HEK 293T cells transfected with S-tagged versions of mouse USP18 (mUSP18), mouse USP18 with the active site cysteine replaced by alanine (mUSP18 C61A), human USP21 (hUSP21), zebrafish USP18 ( dr USP18) and zebrafish USP18 with the active site cysteine replaced by an alanine ( dr USP18 C38A) or from untransfected cells (control). Protein expression was visualized with an antibody directed against the S tag. ( b ) Immunoblot of lysates from cells transfected with the indicated expression constructs, incubated with the active site–directed probe ISG15-PA. Complex formation was monitored on the basis of a size shift detected with an anti-S-tag antibody. ( c ) Immunoblot analyses of protein lysates from cells transfected with the indicated expression constructs, incubated with the active site–directed probe Ub-PA. Complex formation was monitored on the basis of a size shift detected with an anti-S-tag antibody. USP21 is cross-reactive for ISG15, and ubiquitin served as a positive control for Ub binding. Results shown in a – c are representative of three independent experiments.

Article Snippet: The vector pTriEx2-USP21cd encoding residues 197–565 of human USP21 was generated by amplification of USP21 cDNA from the vector Flag-HA-USP21 (Addgene) with the primers KpnI-USP21cd-for and XhoI-USP21cd-rev.

Techniques: Western Blot, Transfection, Expressing, Construct, Incubation, Positive Control, Binding Assay