This week we mainly focused on the selection and discussion of topics (excluding preliminary social research and expert interviews).
This week's focus is on gene cloning, or in other words, plasmid construction and validation.
We optimized the codon and cloned the ADH3 gene (removed signal peptide sequence) to the pET46/Ek-LIC vector to construct the recombinant plasmid pET46/ADH3.
In the past fortnight, we has mainly focused on protein characterization and homogeneity analysis.
In the past half month, we has focused on the structural analysis of cryo-EM, which referred to the sample preparation and data collection, data processing and 3D reconstruction, model construction and space optimization.
This week we focused on structural analysis. We conducted a detailed analysis of substrate binding pockets, such as pocket composition and interaction, substrate binding key residues, OTA binding patterns, etc.
This week we focused on rational design strategies for conceptualizing mutants.
Based on the ADH3-D344N/OTA complex structure, we designed the amino acids bound to the substrate OTA to inactivate them one by one to verify their catalytic activity against OTA.
This week we mainly focused on fixed-point mutation experiments. We successfully constructed mutant S88A,H163A,K210A,V217A,L218A,H251A,H253A,I325A and V347A.
In the past fortnight, we has mainly focused on the analysis and comparison of mutant enzyme active substances.
After protein expression and purification, a SDS-PAGE was carried out to verify whether the protein had been expressed and the enzyme activity assay showed the key amino acid function, and we proposed the catalytic mechanism model of ADH3.
This week we focused on the rational design strategies for conceptualizing mutants.
Based on the results of the previous round of mutations, we designed mutations for L218, V347, and S88 sites:
In the past fortnight, the focus has been on mutant protein expression and purification.
The mutants were constructed and expressed, and the purity was verified through SDS-PAGE, showing that the protein purity was over 95% and the molecular weight was about 45 kDa.
In the past fortnight, we has mainly focused on the analysis and comparison of mutant enzyme active substances.
The activities of S88E, S88K and S88H were increased by 3.7, 2.4 and 1.3 times, respectively. The activity of the series L218 and V347 mutants was significantly reduced.
In the past fortnight, we has mainly focused on the review of the topic and analyzed the reasons for errors in the experiment as a whole and discussed the follow-up development direction of the topic.
This week's focus was on the bioinformatics analysis, i.e. sequence analysis and genetic design.
Based on the sequence analysis in the NCBI database, we compared AHD3 to screen the optimal gene sequence of interest (LlADH). Comprehensive analysis through bioinformatics tools. At the same time, its structural domains are predicted and compared.
This week's focus was on the gene cloning, which referred to the plasmid construction and validation.
We optimized the codon and cloned the LlADH gene (removed signal peptide sequence) to the pET46/Ek-LIC vector to construct the recombinant plasmid pET46/LlADH. We obtained 8 sequence-correct clones and saved them as LlADH-WT-1~8. Purification verification was performed at the same time.
In the past fortnight, we has mainly focused on protein characterization and homogeneity analysis.
This week's focus was on the analysis of LlADH hydrolysates and the acquisition of standard sample data.
In the past fortnight, we has mainly focused on the analysis and comparison of enzyme active substances.
We also compared the activity of LlADH with ADH3, and found that the hydrolytic activity of LlADH against OTA was twice that of ADH3.
In the past fortnight, the focus has been on the structural analysis of cryo-EM. That is, sample preparation and data collection, data processing and 3D reconstruction, model construction and space optimization.
This week we focused on the structural analysis and rational design.
We conducted a detailed analysis of substrate-bound pockets, such as pocket composition and interaction, substrate binding to key residues, OTA binding mode (based on ADH3 superposition), etc. At the same time, the structural differences between LlADH and ADH3 were compared in detail.
This week we focused on rational design strategies for conceptualizing mutants.
Based on the ADH3/OTA complex structure, we designed mutants targeting substrate-binding pocket key residues. A total of 15 mutation types were designed.
This week we mainly focused on fixed-point mutation experiments.
The mutants we successfully constructed are S89D, S89F, S89H, S89K, and S89R; S91R; G131L; L219H; I326A; V348F,V348H,V348S.
Some mutants failed to be built, such as G131F, L219K, and L219R.
This week we focused on small-scale protein expression testing. We selected strains E. coli BL21 (DE3) to optimized the conditions for expression by testing.
In the past fortnight, we has mainly focused on large-scale protein expression and purification.
The purity verification by SDS-PAGE showed that the protein was over 95% and the molecular weight was about 45 kDa.
In the past fortnight, we has mainly focused on the analysis and comparison of mutant enzyme active substances.
Based on wild type, the relative activity was 100%, and the activity of mutant I326A was significantly increased. V348F activity slightly increased; S89D, S89H, S89K, S89R, S91R, G131L, V348S, V348H activity decreased; L219H was completely inactivated.
In the past fortnight, we has mainly focused on the review of the topic and analyzed the reasons for errors in the experiment as a whole and discussed the follow-up development direction of the topic.
In the past fortnight, the main focus has been on the upgrading of the expression system of mutants. We achieved efficient expression in Pichia pastoris, laying the foundation for industrialization and practical application.