SpSrtA-Catalyzed Isopeptide Ligation on Lysine Residues
Sortase-mediated ligation (SML) is widely used for protein bioconjugation. However, the sortase used in this strategy typically recognizes only the N-terminal oligoglycine, which is absent in most natural proteins. To broaden the spectrum of substrates compatible with SML, we focus on a novel sortas...
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| Format: | Article |
| Language: | English |
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MDPI AG
2024-01-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/12/1/179 |
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| author | Jiajia Wu Tianyu Chu Jian Hao Liang Lin |
| author_facet | Jiajia Wu Tianyu Chu Jian Hao Liang Lin |
| author_sort | Jiajia Wu |
| collection | DOAJ |
| description | Sortase-mediated ligation (SML) is widely used for protein bioconjugation. However, the sortase used in this strategy typically recognizes only the N-terminal oligoglycine, which is absent in most natural proteins. To broaden the spectrum of substrates compatible with SML, we focus on a novel sortase, sortase A from <i>Streptococcus pneumoniae</i> (SpSrtA), known for its expanded substrate specificity (N-terminal glycine, alanine, and serine). We present the first evidence showing that the reported SpSrtA mutant (SpSrtA*) can modify lysine residues in itself and other proteins. The modification sites of SpSrtA* were identified through LC-MS/MS analysis. Moreover, we discovered an optimal lysine-containing peptide tag by fusing it onto sfGFP, resulting in a labeling efficiency of 57%. Inspired by this, we applied the method to modify proteins on microorganism surfaces up to 13.5-fold. To enhance labeling efficiency, we fused the SpSrtA* onto a surface protein and achieved a 2.64-fold improvement. We further developed a high-throughput yeast display screening method for the directed evolution of SpSrtA*, achieving a 10-fold improvement in the labeling efficiency of this surface protein. Our study provides a novel strategy for modifying the lysine residues that will be a powerful addition to the protein bioconjugation toolbox. |
| first_indexed | 2024-03-08T09:49:45Z |
| format | Article |
| id | doaj.art-74bf864ddbef4b8298e9e4f9fe4edc66 |
| institution | Directory Open Access Journal |
| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-08T09:49:45Z |
| publishDate | 2024-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Microorganisms |
| spelling | doaj.art-74bf864ddbef4b8298e9e4f9fe4edc662024-01-29T14:06:49ZengMDPI AGMicroorganisms2076-26072024-01-0112117910.3390/microorganisms12010179SpSrtA-Catalyzed Isopeptide Ligation on Lysine ResiduesJiajia Wu0Tianyu Chu1Jian Hao2Liang Lin3Department of Chemistry, Shanghai University, Shanghai 200444, ChinaCollege of Chemistry and Molecular Engineering, Peking University, Beijing 100871, ChinaDepartment of Chemistry, Shanghai University, Shanghai 200444, ChinaState Key Laboratory of Chemical Biology, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, ChinaSortase-mediated ligation (SML) is widely used for protein bioconjugation. However, the sortase used in this strategy typically recognizes only the N-terminal oligoglycine, which is absent in most natural proteins. To broaden the spectrum of substrates compatible with SML, we focus on a novel sortase, sortase A from <i>Streptococcus pneumoniae</i> (SpSrtA), known for its expanded substrate specificity (N-terminal glycine, alanine, and serine). We present the first evidence showing that the reported SpSrtA mutant (SpSrtA*) can modify lysine residues in itself and other proteins. The modification sites of SpSrtA* were identified through LC-MS/MS analysis. Moreover, we discovered an optimal lysine-containing peptide tag by fusing it onto sfGFP, resulting in a labeling efficiency of 57%. Inspired by this, we applied the method to modify proteins on microorganism surfaces up to 13.5-fold. To enhance labeling efficiency, we fused the SpSrtA* onto a surface protein and achieved a 2.64-fold improvement. We further developed a high-throughput yeast display screening method for the directed evolution of SpSrtA*, achieving a 10-fold improvement in the labeling efficiency of this surface protein. Our study provides a novel strategy for modifying the lysine residues that will be a powerful addition to the protein bioconjugation toolbox.https://www.mdpi.com/2076-2607/12/1/179isopeptide ligationsortase Ayeast display |
| spellingShingle | Jiajia Wu Tianyu Chu Jian Hao Liang Lin SpSrtA-Catalyzed Isopeptide Ligation on Lysine Residues Microorganisms isopeptide ligation sortase A yeast display |
| title | SpSrtA-Catalyzed Isopeptide Ligation on Lysine Residues |
| title_full | SpSrtA-Catalyzed Isopeptide Ligation on Lysine Residues |
| title_fullStr | SpSrtA-Catalyzed Isopeptide Ligation on Lysine Residues |
| title_full_unstemmed | SpSrtA-Catalyzed Isopeptide Ligation on Lysine Residues |
| title_short | SpSrtA-Catalyzed Isopeptide Ligation on Lysine Residues |
| title_sort | spsrta catalyzed isopeptide ligation on lysine residues |
| topic | isopeptide ligation sortase A yeast display |
| url | https://www.mdpi.com/2076-2607/12/1/179 |
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