Staphylococcus aureus sortase a high efficiency mutant

A staphylococcus and mutant technology, applied in the field of bioengineering, can solve problems such as low catalytic efficiency, and achieve the effect of improving catalytic efficiency and efficiency
CN106191015BActive Publication Date: 2019-09-13PEKING UNIV
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
PEKING UNIV
Publication Date
2019-09-13

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Abstract

The invention discloses a highly efficient mutant of staphylococcus aureus sortase A. Starting from the Staphylococcus aureus sortase A gene and a known mutant gene, a mutant library was constructed by error-prone PCR and site saturation mutation. Using a screening platform based on fluorescence resonance energy transfer, after multiple rounds of screening and mutation A series of sortase A mutants with improved catalytic activity comprising D124G, Y187L, E189R and / or F200L mutations were obtained by integration. These sortase A mutants can not only improve the efficiency of catalyzing the reaction between LPXTG and the atypical substrate α-Gly, but also improve the efficiency of catalyzing the reaction between LPXTG and the typical substrate α-Gly n The efficiency of the reaction between.
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Description

technical field

[0001] The present invention relates to the directed evolution of protease in the field of bioengineering, in particular to the evolution of a mutant of Staphylococcus aureus sortase A by constructing a high-throughput screening platform, which can efficiently catalyze LPXTG and α-Gly or α- Gly n Linkage between reactions. Background technique

[0002] Staphylococcus aureus sortase A (Sa-SrtA) is a class of cysteine ​​transpeptidase ubiquitous in Gram-positive bacteria. Typically, it can catalyze the substrate LPXTG (X represents any amino acid) and α -Gly n The ligation reaction between (oligoglycine), this reaction is therefore named sortase-mediated ligation (Sortase-Mediated Ligation, SML). At the same time, sortase A can also catalyze the reaction between LPXTG and α-Gly, however, compared to its catalyzed α-Gly n reaction, the reactivity is lower.

[0003] Using the sortase-mediated ligation reaction, the site-directed labeling and coupling of vari...

Claims

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