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

Active Publication Date: 2019-09-13
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low catalytic efficiency of wild-type sortase A, it is necessary to improve the activity of sortase A by directed evolution based on high-throughput screening and rational design.

Method used

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  • Staphylococcus aureus sortase a high efficiency mutant
  • Staphylococcus aureus sortase a high efficiency mutant
  • Staphylococcus aureus sortase a high efficiency mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1. Construction of Staphylococcus aureus sortase A random mutation library using error-prone PCR

[0035] Using error-prone PCR to introduce nucleotide mutations into the Staphylococcus aureus sortase A gene in vitro, the specific conditions for error-prone PCR are as follows:

[0036]

[0037]

[0038]The PCR template is a plasmid containing the wild-type Staphylococcus aureus sortase A gene. Firstly, the nucleotide sequence corresponding to the amino acid sequence from the 26th to the 206th position is cloned into between the NdeI and XhoI restriction sites of the pet28a vector by subcloning.

[0039] Upstream primer: 5'-GGAATTCCATATGAAACCACATATCGATAATTATC-3' (SEQ ID NO.11)

[0040] Downstream primer: 5'-GGTAGGCACTCGAGTTATTTGACTTCTGTAGCTAC-3' (SEQ ID NO.12)

[0041] PCR amplification conditions: 95°C for 5 min; 95°C for 1 min, 55°C for 1 min, 72°C for 1 min, 30 cycles; 72°C for 10 min.

[0042] The error-prone PCR amplification product was purified by...

Embodiment 2

[0043] Example 2, Construction of Staphylococcus aureus sortase A site saturation mutation library by point mutation PCR

[0044] Use the point mutation PCR method to introduce saturation mutations into the two sites Tyr187 and Glu189 of the P94R / D160N / D165A / K190E / K196T mutant of sortase A, and the PCR conditions are as follows:

[0045]

[0046]

[0047] The PCR template is the gene of sortase 2, which is obtained by mutating the pet28a plasmid containing the wild-type sortase A gene in Example 1.

[0048] Upstream primer: 5'-CATTAATTACTTGTGATGATNNKAATNNKGAGACAGGCGTTTGGG-3' (SEQ ID NO.13)

[0049] Downstream primer: 5'-CCCAAACGCCTGTCTCMNNATTMNNATCATCACAAGTAATTAATG-3' (SEQ ID NO.14)

[0050] In the above primers, N represents one of the four bases A, C, G, and T, K represents G or T, and M represents A or C.

[0051] PCR amplification conditions: 95°C for 5min; 95°C for 45s, 55°C for 45s, 72°C for 7min, 23 cycles; 72°C for 10min.

[0052] The point mutation PCR produc...

Embodiment 3

[0053] Example 3. Construction and verification of a screening system based on fluorescence resonance energy transfer

[0054] Using the method of subcloning, the fluorescent proteins of eGFP-LPETG and G-cpV were fused and expressed on the pet28a vector, purified using 6×His tags, and the purified proteins were subjected to a sortase-mediated ligation reaction, such as figure 1 As shown, EGFP and cpV proteins are suitable donors and acceptors of fluorescence resonance energy transfer (FRET). The fluorescence resonance energy transfer efficiency between proteins is improved, and the higher the sortase activity is, the higher the fluorescence function energy transfer efficiency is. Validation of the screening platform using wild-type sortase A and departure sortase 2 demonstrated that sortase A activity is directly proportional to fluorescence resonance energy transfer efficiency, as figure 2 shown.

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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.

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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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/52C12N15/55C12P21/00
CPCC12N9/52C12P21/00C12Y304/2207
Inventor 陈鹏陈龙
Owner PEKING UNIV
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