Journal:

Article Title: Inhibition of Filovirus Replication by the Zinc Finger Antiviral Protein ▿

doi: 10.1128/JVI.01601-06

Figure Lengend Snippet: Inhibition of Zaire-EBOV replicon system by ZAP. Huh-T7 cells were transfected with plasmids encoding Zaire-EBOV NP, L protein, VP35, VP30, and minigenome containing a Renilla luciferase gene. Increasing amounts of plasmid encoding ZAP-HA were cotransfected as indicated (−ctrl, negative control without L plasmid; +ctrl, positive control without ZAP-HA plasmid). Empty vector pTM1 was used to keep the amount of transfected DNA constant. Plasmid pTM1-FFluc was included to normalize Renilla luciferase values. Luciferase activities were determined 2 days posttransfection. The expression levels of ZAP-HA and EBOV NP were analyzed by Western blot using anti-HA monoclonal antibody and anti-Zaire-EBOV polyclonal antibody, respectively (bottom). The amount of cell lysate loaded on the protein gel was adjusted according to the firefly luciferase values to ensure comparability with the normalized Renilla values. The levels of GAPDH served as a loading control.

Article Snippet: To generate an α- 32 P-labeled riboprobe, 2 μl of the DNA template was used in a 20-μl in vitro transcription reaction mixture containing 1× transcription buffer, 25 U of T7 DNA-dependent RNA polymerase (New England BioLabs), 16 U RNaseOUT, 500 μM each of 5 mM ATP, 5 mM GTP, and 5 mM UTP, and 5 μl of [α- 32 P]CTP (15 TBq/mmol; Hartmann Analytic GmbH).

Techniques: Inhibition, Transfection, Luciferase, Plasmid Preparation, Negative Control, Positive Control, Expressing, Western Blot

Journal:

Article Title: Inhibition of Filovirus Replication by the Zinc Finger Antiviral Protein ▿

doi: 10.1128/JVI.01601-06

Figure Lengend Snippet: Effect of L-protein overexpression on ZAP-induced inhibition of the Zaire-EBOV replicon system. (A) Huh-T7 cells were transfected with plasmids encoding Zaire-EBOV NP, VP35, VP30, increasing amounts of L-protein expression plasmid, and a minigenome containing a Renilla luciferase gene (−ctrl, negative control without L plasmid). Different amounts of ZAP-HA expression plasmid were cotransfected. Empty vector pTM1 was used to keep the amount of transfected DNA constant. Plasmid pTM1-FFluc was included to normalize Renilla luciferase values. (B) Expression levels of ZAP-HA and EBOV NP were analyzed by Western blot using anti-HA monoclonal antibody and anti-Zaire-EBOV polyclonal antibody, respectively. The amount of cell lysate loaded on the protein gel was adjusted according to the firefly luciferase values to ensure comparability with the normalized Renilla values. The levels of GAPDH served as a loading control.

Article Snippet: To generate an α- 32 P-labeled riboprobe, 2 μl of the DNA template was used in a 20-μl in vitro transcription reaction mixture containing 1× transcription buffer, 25 U of T7 DNA-dependent RNA polymerase (New England BioLabs), 16 U RNaseOUT, 500 μM each of 5 mM ATP, 5 mM GTP, and 5 mM UTP, and 5 μl of [α- 32 P]CTP (15 TBq/mmol; Hartmann Analytic GmbH).

Techniques: Over Expression, Inhibition, Transfection, Expressing, Plasmid Preparation, Luciferase, Negative Control, Western Blot

Journal: Science Advances

Article Title: A general temperature-guided language model to design proteins of enhanced stability and activity

doi: 10.1126/sciadv.adr2641

Figure Lengend Snippet: The structures and experimental results of single-site mutants predicted by PRIME for LbCas12a ( A and B ), T7 RNA polymerase ( C and D ), creatinase ( E and F ), nonnatural nucleic acid polymerase ( G and H ) and VHH ( I and J ) are depicted. The data points representing the mutations were systematically arranged in ascending order, with the corresponding value for the wild-type protein delineated by a gray bar for comparative purposes. Mutants that exhibited superior performance compared to their wild-type counterparts in terms of targeted attributes are highlighted in yellow, while negative mutants are shown in blue. The engineering goals varied between proteins for practical purposes: for LbCas12a, T7 RNA polymerase, and creatinase, the objective was enhanced thermostability ( T m ); for nonnatural nucleic acid polymerase (Tgo-D4K), the aim was to accelerate the synthesis rate of FANA; and for VHH, the goal was to improve the tolerance ability under extreme alkaline pH conditions [median effective concentration (EC 50 ) of VHH binding to the antigen]. All mutated structure were folded by Alphafold2. Detailed experimental data can be found in the separate Excel file in the Supplementary Materials.

Article Snippet: However, compared to the commercial thermostable T7 RNA polymerase (Hi-T7, T m = 56.8°C) available from New England Biolabs, our eight-site mutant still has a T m that is 4°C lower.

Techniques: Concentration Assay, Binding Assay

Journal: Science Advances

Article Title: A general temperature-guided language model to design proteins of enhanced stability and activity

doi: 10.1126/sciadv.adr2641

Figure Lengend Snippet: ( A ) Comparative web-lab results for the top 15 single-point mutations in T7 RNA polymerase. ( B ) Results for Tgo-D4K, as determined by PRIME, Rosetta, ESM-vote, and ESM2(homo). ( C and D ) Maximum (C) and mean (D) fitness outcomes obtained from in silico–directed evolution on the GB1 dataset, involving random mutagenesis, ftMLDE, ESM-2, and PRIME. For ESM-2 and PRIME, we examined both top- K sampling and the tiered sampling used by ftMLDE .

Article Snippet: However, compared to the commercial thermostable T7 RNA polymerase (Hi-T7, T m = 56.8°C) available from New England Biolabs, our eight-site mutant still has a T m that is 4°C lower.

Techniques: In Silico, Mutagenesis, Sampling

Journal: Science Advances

Article Title: A general temperature-guided language model to design proteins of enhanced stability and activity

doi: 10.1126/sciadv.adr2641

Figure Lengend Snippet: The protein structures and experimental results for multisite mutants of LbCas12a (predicted by Alphafold2) ( A and C ) and T7 RNA polymerase (PDB ID: 1MSW) ( B and D ). The functional domains of each protein are depicted beneath their structural diagrams. In (C) and (D), a red dashed line indicates the normalized activity level of the wild type. The activities of the five most thermostable mutants are marked with red dots to facilitate direct comparison with the wild type. The thermal stability ( T m ) of the wild type is represented by a yellow dashed line for LbCas12a and a blue dashed line for T7 RNA polymerase. R1 and R2 represent the first and the second rounds of multipoint combination, respectively, with the numbers following them indicating the mutant indices.

Article Snippet: However, compared to the commercial thermostable T7 RNA polymerase (Hi-T7, T m = 56.8°C) available from New England Biolabs, our eight-site mutant still has a T m that is 4°C lower.

Techniques: Functional Assay, Activity Assay, Comparison, Mutagenesis