1). Transformation and verification of the wild-type (WT) pET-47b(+)-NT-HRV3CP plasmid.
2). Induced expression of the WT NT*-HRV3CP protein.
3). Preliminary purification of the WT NT*-HRV3CP protein.
4). Buffer exchange and concentration of the purified protein.
1). Construction of NT*-HRV3CP mutant plasmids via site-directed mutagenesis.
2). Sequencing verification of the mutant plasmids.
3). Induced expression of the mutant proteins.
3). Induced expression of the mutant proteins.
4). Purification and post-processing of the mutant proteins.
1). Establishment of the enzymatic reaction system for both WT and mutant proteins.
2). Setup and incubation of hydrolysis and ligation reaction groups.
3). Development of the HPLC method and analysis of samples.
4). Experimental data analysis and project summary.
| Date | Members Responsible | Experimental Content | Results |
|---|---|---|---|
| July 4-31 | Xiaoken Lin, Yijie Wang, Liya Ma, Yuhang Sheng | Expression and purification of the wild-type (WT) NT*-HRV3CP protein. | High-purity WT protein was successfully obtained for subsequent reactions. |
| Aug. 1-15 | Xiaoken Lin, Yuhang Sheng, Heming Li, Liya Ma | Construction of NT*-HRV3CP mutant plasmids. | All target mutant plasmids were successfully constructed and verified by Sanger sequencing. |
| Aug. 16-31 | Xiaoken Lin, Yuhang Sheng, Heming Li | Expression and purification of NT*-HRV3CP mutant proteins. | High-purity versions of each mutant protein were successfully obtained. |
| Sep. 1-30 | Xiaoken Lin, Yuhang Sheng, Heming Li, Yijie Wang | Enzymatic activity assay and HPLC analysis of WT and mutant proteins. | Comparative enzymatic activity data for all proteins were obtained, clarifying the impact of key residues. |
This
study aimed to convert the HRV-3C protease, a highly specific cysteine hydrolase, into a novel
peptide ligase through rational design. Our primary strategy involved computationally engineering
a hydrophobic active-site pocket to suppress the enzyme’s intrinsic hydrolytic activity by sterically
hindering the access of nucleophilic water molecules.